Review
mRNA quality control pathways in Saccharomyces cerevisiae
SATARUPA DAS and BISWADIP DAS*
Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India
*Corresponding author (Fax, +91-33-2414-6414; Email, [email protected])
Efficient production of translation-competent mRNAs involves processing and modification events both in the
nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and posttranscriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant nonfunctional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of
mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional
counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly
result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To
prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes
to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role
of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.
[Das S and Das B 2013 mRNA quality control pathways in Saccharomyces cerevisiae. J. Biosci. 38 615–640] DOI 10.1007/s12038-013-9337-4
1.
mRNA biogenesis in eukaryotic cell
Productive expression of an eukaryotic gene involves the
synthesis of its message, its subsequent maturation events in
the nucleus, export of the matured message from the nucleus to
cytoplasm, structural remodelling of the exported message to
adopt a translation competent state, translation of the competent message and finally its destruction in the specific location
in the cytoplasm (Le Hir et al. 2001; Jensen et al. 2003; Stutz
and Izaurrelde 2003; Vinciguerra and Stutz 2004; Kohler and
Hurt 2007; Luna et al. 2008). During this long journey, mRNA
undergoes extensive structural modification and remodelling
events both in the nucleus as well as in the cytoplasm. Nuclear
events of mRNA biogenesis advances through the transcription of a given gene by RNA polymerase II followed by the
capping of the nascent message at 5′-end, splicing to remove
intron(s) and maturation towards the 3′-end of the message
involving a site-specific cleavage and template-independent
polyadenylation (Proudfoot et al. 2002) (figure 1). In addition
to these covalent modification events, the maturing mRNAs
during all of these steps become associated with a wide repertoire of mRNA-maturing factors and heterogeneous nuclear
Keywords.
ribonucleoproteins (hnRNPs) and finally get packaged into
properly conformed mature mRNPs (Le Hir et al. 2001;
Jensen et al. 2003; Stutz and Izaurrelde 2003; Vinciguerra
and Stutz 2004; Kohler and Hurt 2007; Luna et al. 2008).
For example, a heterodimeric nuclear cap-binding complex
(CBC) consisting of a 80 kDa protein Cbc1p and a 20 kDa
subunit Cbc2p binds to the 5′-cap of the nascent mRNA when
the transcript length is just 20–30 nt (Izaurralde et al. 1992,
1994). Secondly, the transcription/export (TREX) complex
consisting of THO complex (Hpr1p, Mft1p, Tho2p, Thp2p)
and mRNA export factor RNA helicase Sub2p (known as
UAP56 in human); RNA-binding protein Yra1p (REF/ALY
in human) are deposited onto the transcribing and growing
mRNAs in a transcription-dependent manner. This is followed
by the binding of the maturing mRNP with export receptor
Mex67p:Mtr2p (NXF1:p15 in human), many heterogeneous
nuclear ribonucleoproteins (hnRNPs) (such as Gbp1p, Hrb1p
and Tex1p) and poly(A) tail-binding protein Pab1p to achieve
properly formed export competent mRNPs in the nucleus
(figure 1) (Le Hir et al. 2001; Jensen et al. 2003; Stutz and
Izaurrelde 2003; Vinciguerra and Stutz 2004; Kohler and Hurt
2007; Luna et al. 2008). These export-competent mRNPs are
Cbc1p; DRN; exosome; mRNA decay; mRNA surveillance; NMD; quality control; Rrp6p
Abbreviations used: CBC, cap-binding complex; DRN, decay of mRNA in the nucleus; NGD, no-go delay; NMD, nonsense-mediated
decay; NSD, non-stop decay; PTC, premature termination codon
http://www.ias.ac.in/jbiosci
Published online: 10 July 2013
J. Biosci. 38(3), September 2013, 615–640, * Indian Academy of Sciences
615
616
Satarupa Das and Biswadip Das
DNA
Transcription
THO Components
Pre-mRNA
UAA
Export factor Sub2p/
Yra1p/Mex67p
capping
Exon Junction Complex
Poly(A) Binding Protein
Pab1p
Cbc1P m7Gppp
Capped mRNA
UAA
Splicing
Ribosome
Cbc1P m7Gppp
Spliced mRNA
UAA
Polyadenylation
NUCLEUS
Cbc1P m7Gppp
UAA
AAAAAAAA70
Export
CYTOPLASM
Shuttling of Released
Export Factors to Nucleus
Cbc1P
or
Polyadenylated
and Export
Competent mRNA
m7Gppp
A
AAAAAAAA70
Mature Translating mRNA
4E
Translation
Degradation
Figure 1. mRNA life-cycle in eukaryotic cell: Schematic view of the nuclear and cytoplasmic phases of mRNA life cycle, namely
transcription, capping, splicing, polyadenylation, nuclear export, translation and finally degradation in cytoplasm. Various mRNA binding
proteins which are deposited onto/remain associated with maturing transcripts during different stages of nuclear phase of life are shown by
colored solid symbols on the transcript body. Each symbol is either annotated directly in the diagram or denoted in the legend box. Note that
THO components/maturing factors/mRNA-binding proteins are released from mRNA once the mRNA matures and become export
competent. Similarly export factors are also released from transcript body once mRNA arrives at the cytoplasm and finally shuttle back
to nucleus. In the cytoplasm, messenger RNAs may remain associated either with nuclear CBC (while undergoing pioneer round of
translation) or with eIF4E (while undergoing subsequent steady state translation) which is indicated in the cytoplasmic phase of the
diagram. Also for simplicity other mRNA binding proteins which remain associated to translating mRNAs in the cytoplasm are not shown
except for cap-binding complex (CBC), translation initiation factor 4E. AUG and UAA indicate the beginning and end of the open reading
frame (ORF) carried by the message. All vertical downward arrowheads represent various biogenesis steps as mentioned in the figure.
then released from the transcription site and gradually move to
nuclear periphery where they become associated with nuclear
pore complexes (NPCs) to be exported from nucleus to cytoplasm (Reed 2003; Dimaano and Ullman 2004) (figure 1).
Thus, nuclear export represents the culmination of nuclear
phase of gene expression that takes place through the NPC
(Tran and Wente 2006; Rodriguez-Navarro and Hurt 2011).
Once in the cytoplasm, the mRNPs undergo the first
round of translation (referred to as the pioneer round of
translation) while they are still bound to nuclear CBC
(Ishigaki et al. 2001; Gao et al. 2005). Following the pioneer
round, the mRNPs undergo a major structural remodelling
event that exchanges nuclear CBC at the 5′-cap of mRNA
with the translation initiation factors eIF4E/eIF4G (Maquat
et al. 2010). These remodelled messages finally engage in
the steady state of productive translation to produce the total
J. Biosci. 38(3), September 2013
cellular pool of proteins (Ishigaki et al. 2001; Gao et al.
2005). Some of the exported mRNPs may also be
transported, localized and confiscated in several specific
cytoplasmic locations (such as in the stress granules) for
future use (Novar et al. 1983; Kedersha et al. 1999).
Finally, once a specific message has completed its cellular
duty it is usually destroyed by the default degradation pathway (see below in section 2) at a specified cytoplasmic
location, known as P-bodies in Saccharomyces cerevisiae
(Sheth and Parker 2006).
In yeast Saccharomyces cerevisiae, transcription and premRNA processing steps in the nucleus are physically and
functionally coupled with the C-terminal domain (CTD) of
the largest subunit Rpb1p, of the RNA polymerase II
(Neugebauer 2002; Buratowski 2009). The CTD acts as a
loading platform for several transcription and other mRNA
mRNA quality control pathways in Saccharomyces cerevisiae
processing factors such as splicing, export factors and the
proteins involved in the formation of matured 3′-end of the
message (Maniatis and Reed 2002; Reed 2003; Rodriguez
et al. 2004; Aguilera 2005; Bentley 2005; Bird et al. 2005;
Reed and Cheng 2005). Each of these mRNA processing
steps physically communicates with each other and imparts
either a stimulatory or an inhibitory influence depending on
the status of the previous processing event(s) (Maniatis and
Reed 2002; Reed 2003; Rodriguez et al. 2004; Aguilera
2005; Bentley 2005; Bird et al. 2005; Reed and Cheng
2005). Although partly mediated by the phosphorylation
status of the specific serine residues in the c-terminal domain
of Rpb1p subunit of RNA polymerase II enzyme, the exact
molecular mechanism of this communication is not very
clear. Nevertheless, it has been demonstrated that functional
coupling (i) enhances the efficiency and likelihood of production of export competent and productive mRNPs and (ii)
minimizes the possibility of formation of functionally defective transcripts, thus lowering the risk of formation of
unproductive polypeptides as well as reducing the requirement of quality control (Rodriguez et al. 2004; Aguilera
2005; Bentley 2005; Bird et al. 2005; Reed and Cheng
2005). In spite of tight coupling, defective/faulty messages
still arise, which are rapidly eliminated by a varieties of
surveillance and quality control mechanisms (see below).
2.
mRNA degradation pathways in Saccharomyces
cerevisiae
For a long time mRNA degradation was viewed as an
uncontrolled activity of the cell that was a nuisance and
threat to experiments associated with messenger RNAs in
general. However, during last two decades it became clear
that mRNA decay, like any other aspects of mRNA metabolism, is also a highly regulated process, precisely controlled
by distinct set of genes (Beelman and Parker 1994;
Caponigro and Parker 1996; Parker 2012). For example,
mRNA decay had been shown to determine the basal level
of cellular RNA expression and its regulated variation imposed by environmental stimuli (reviewed in Parker 2012).
Second, mRNA decay was demonstrated to play a crucial
role in limiting the formation of defective and aberrant
mRNAs via their selective decay which would otherwise
have generated deleterious polypeptides (reviewed in
Fasken and Corbett 2005; Houseley and Tollervey 2006;
Doma and Parker 2007; Parker 2012). In addition, RNA
degradation was known to remove byproducts of gene expressions such as excised introns, external or internal
spacers, etc. (reviewed in Houseley and Tollervey 2006;
Doma and Parker 2007; Parker 2012). Finally, RNA decay
mechanisms degrade other intragenic, intergenic, promoterassociated and anti-sense RNAs, which are produced either
as regulatory RNAs or considered as transcriptional noise
617
(Davis and Ares 2006; Wyers et al. 2005; Neil et al. 2009;
Xu et al. 2009).
Broadly, mRNA degradation systems are classified into two
kinds: (a) general default decay pathways and (b) specialized
mRNA decay pathways. Default decay pathways are involved
in the maintenance of general cellular turnover of all mRNAs
after they perform their pre-requisite cellular functions
(Beelman and Parker 1994; Caponigro and Parker 1996;
McCarthy 1998). Default decay is initiated by the process of
deadenylation, which is usually carried out by two complexes
in Saccharomyces cerevisiae, namely, Ccr4p/Pop2p/Not complex (major deadenylation machinery) and the Pan2p/Pan3p
complex (a subsidiary deadenylation machinery) (figure 2;
table 1). Deadenylation in Saccharomyces cerevisiae typically
involves the sequential removal of adenylate residues from the
poly(A) tail to convert it into an oligo-A state containing 10–
15 adenylate residues. This is followed by the removal of
5′-cap structure by the concerted action of Dcp1p/Dcp2p (called
decapping complex) (Schwartz and Parker 2000; She et al.
2004; reviewed in Parker 2012), which in turn is followed by
the degradation of the transcript body either in 5′→3′ direction
by major cytoplasmic exoribonuclease Xrn1p (Poole and
Stevens 1995). Alternatively, mRNA can also be degraded in
3′→5′ direction by cytoplasmic exosome and Ski complex
following deadenylation independent of decapping (reviewed
in Doma and Parker 2007; Parker 2012) (figure 2; table1).
Exosome is a large multiprotein complex consisting of about
ten or more 3′-5′ exoribonuclease-like subunits that is involved
in the processing and degradation of multitudes of RNA substrates (section 6.1) (Lykke-Andersen et al. 2011; Chlebowski
et al. 2013). After pre-requisite rounds of translation, most of
the normal mRNAs are degraded in the cytoplasm by default
decay system.
Interestingly, several recent studies have provided very
strong evidences that rate of synthesis and degradation of
several mRNAs by default decay pathway within the cell
may be integrated. These studies demonstrated that the promoter and associated cis-regulatory elements of these genes
and some of the transcription factors binding them may regulate the stability and decay rates of their corresponding messages (Enssle et al. 1993; Lotan et al. 2005, 2007;
Harel-Sharvit et al. 2010; Dori-Bachash et al. 2011, 2012;
Bregman et al. 2011; Bellofatto and Wilusz 2011; Trcek
et al. 2011). Promoter swapping experiments showed that
exchanging the native upstream regulatory sequences (UAS)
of RPL30 gene with that of the ACT1 UAS without altering
its coding sequence have remarkable influence on the stability
of RPL30 mRNA (Bregman et al. 2011). The messagecarrying RPL30 coding sequence with the RPL30 UAS
was found to undergo decay very rapidly compared to the
one that has ACT1 UAS. This promoter-influenced decay
was mediated by Rap1p, which was termed as a
‘synthetodegradase’ factor to underscore its effect on both
enhanced transcription and reduced decay under a specified
J. Biosci. 36(3), September 2013
Satarupa Das and Biswadip Das
m7Gppp
AUG
UAA
AAAAAAAA 70
Ccr4p/
Po2/Not
618
Deadenylation
m7Gppp
Dcp1p/
2p
m7Gpp + p
AUG
UAA
3
Decapping
AUG
UAA
A10
m7Gppp
A10
5 Exonucleolytic Decay by exosome
Exosome
AUG
5 3 Exonucleolytic
Decay by Xrn1p
Xrn1p
UAA
A10
Figure 2. Default pathway of mRNA degradation in S. cerevisiae: Almost all mRNAs undergo decay by the deadenylation dependent
pathway. In this mechanism the poly(A) tail is gradually and progressively shortened by a deadenylase activity by Ccr4/Pop2/Not complex.
Following deadenylation, two mechanisms can degrade the mRNA. The major mechanism follows through decapping by Dcp1p/2p
followed by 5′→3′ decay by Xrn1p. The minor mechanism involves 3′→5′ decay by the exosome. AUG and UAA indicate the beginning
and end of the open reading frame (ORF) carried by the message. Only relevant decay components are shown by annotated symbols.
Proteins which remain associated to translating/degrading mRNAs during different stages of decay are not shown.
condition of stimulated gene expression (Bregman et al. 2011).
In another independent study, the promoters of SWI5 and
CLB2 genes were shown to play a strong role in modulating
their corresponding messages in a cell-cycle-dependent
manner by using powerful and sensitive single-cell singlemolecule FISH technique (Trcek et al. 2011). This regulation
involves the mitotic exit network (MEN) kinase Dbf2p and its
interacting partner polo kinase Cdc5p (Trcek et al. 2011) as
Table 1. General factors involved in default mRNA decay pathway
Decay factor
Ccr4p/Pop2p/Not
complex
Pan2p/Pan3p complex
Dcp1p/Dcp2p
Xrn1p
Rat1p
Rai1p
Function
Major deadenylase
Ccr4p-catalytic subunit
Pop2p-ancillary catalytic subunit
Not1p-large scaffolding protein
Not2p–5p, Caf130p, Caf40p-accessory
proteins
Additional deadenylase
Initial trimming of poly(A) tail
Pan2p-catalytic subunit
Pan3p-regulatory subunit
mRNA decapping complex
Dcp2p-catalytic subunit
Dcp1p-stimulatory subunit
Major cytoplasmic 5′ to 3′ exonuclease
Degrades mRNA body from 5′ to 3′
Major nuclear 5′ to 3′ nuclease
Nuclear RNA processing and decay
Interacts with and stimulates Rat1
May function in cap quality control
J. Biosci. 38(3), September 2013
Selected references
Chen et al. (2002), Daugeron et al. (2001);
Tucker et al. (2001, 2002)
Brown et al. (1996); Boeck et al.(1996);
Brown and Sachs (1998)
Schwartz and Parker (2000); She et al.(2004,
2008); Deshmukh et al. (2008)
Poole and Stevens (1995); Van Dijk et al. (2003);
Malys et al. (2004); Johnson (1997); Xiang et al. (2009)
Xue et al. (2000); Xiang et al. (2009); Jiao et al. (2010)
mRNA quality control pathways in Saccharomyces cerevisiae
well as the major cytoplasmic deadenylase Ccr4p/Pop2p/Not
complex (see above). Their finding is consistent with a model
where Dbf2p is recruited to SWI5 and CLB2 promoter and
subsequently loaded onto their corresponding messages and
are carried to the cytoplasm. Once in the cytoplasm, Dbf2p in
association with ancillary factor Dbf20p (assist Dbf2p functions and synthetically lethal with it; Toyn et al. 1991) coordinate the timing of the decay of these messages (Trcek et al.
2011). More recently it was shown that swapping the upstream
cis-regulatory sequences of orthologous genes from two yeast
species affects both mRNA transcription and decay for some
genes (Dori-Bachash et al. 2012). Interestingly, adjacent yeast
genes sharing a common promoter have similar mRNA decay
profile, indicating the existence of a pervasive promotermediated co-ordination between transcription and mRNA decay in yeast. These findings indicated a few interesting
issues. How do promoters and transcription factors that
influence mRNA decay rates leave their marks on the
mRNAs for rest of their life cycle? Second, how is that
mark decoded later in the cytoplasm? As mentioned above,
Trcek et al. (2011) provided strong evidence that Dbf2p and
Dbf20p play a role in forming a cell cycle imprint on SWI5 and
CLB2 mRNA. A clue to the second question came from the
studies by Bregman et al. (2011), who suggested that
promoter-mediated decay of RPL30 mRNA involves the
deadenylase Ccr4p/Pop2p/Not complex and cytoplasmic
exoribonuclease Xrn1p.
Interestingly, Dbf2p is part of a larger interactome of
Ccr4p/Pop2p/Not complex, implying that Dbf2p may play
a role in recruiting the decay components onto the imprinted
message and may even link the processes of initial imprinting the message and later recruitment of decay factors selectively onto the imprinted message. While the exact
molecular mechanism of this promoter-mediated decay of
these transcripts remains unknown, these studies clearly
pointed out that coupling the transcription to decay in inverse manner certainly increases the efficacy of the finetuning of the expression of environmentally induced genes
(Bregman et al. 2011; Bellofatto and Wilusz 2011; DoriBachash et al. 2011, 2012; Trcek et al. 2011). This ‘counteraction’ imposed by the synthetodegradeses-like Rap1p
may play a critical role for the regulation of expression of
many mRNAs whose induction were known to occur in
stepwise response (Shalem et al. 2008; Rabani et al. 2011).
Consistent with this idea it has been found that this coupling
phenomenon is employed by approximately 10% of the
yeast genes which are preserved through evolution (DoriBachash et al. 2011, 2012).
Specialized mRNA decay pathways, in contrast, are very
selective in choosing their mRNA substrates and primarily
degrade various classes of aberrant mRNAs. These aberrant
messages might arise either as a consequence of genomic
mutation or due to inherent inaccuracy associated with transcription and various nuclear events of mRNP biogenesis
619
processes as discussed below (section 4). Hence, specialized
decay pathway ensures the quality control of mRNA biogenesis and the fidelity of gene expression process by limiting
the formation of aberrant and defective messages (Fasken
and Corbett 2005; Doma and Parker 2007; Isken and Maquat
2007; Houseley and Tollervey 2009; Parker 2012).
3.
mRNA quality control: Safeguarding the cells
As mentioned above, recent studies have demonstrated that
in the model organism Saccharomyces cerevisiae, abnormal
or aberrant mRNAs/transcripts that are generated spontaneously undergo very rapid degradation in a highly selective
manner, thereby resulting in a very low level of the faulty
transcripts (Burkard and Butler 2000; Bousquet-Antonelli
et al. 2000; Das et al. 2000, 2006; Torchet et al. 2002;
Doma and Parker 2007; Isken and Maquat 2007). Genetic
suppressor analysis and subsequent molecular studies identified a number of genes and pathways in different cellular
compartments that are involved in the rapid degradation of
these abnormal messages (Burkard and Butler 2000;
Bousquet-Antonelli et al. 2000; Das et al. 2000, 2006;
Torchet et al. 2002; Fasken and Corbett 2005 Doma and
Parker 2007; Isken and Maquat 2007). Identification of
Genes was made from the general findings where inactivation/knocking down specific gene(s) resulted in the stabilization of various kinds of aberrant messages. Collective
findings from these studies led to the general conclusion that
eukaryotic cells are equipped with a number of quality
control/surveillance systems that constantly monitor the various events of mRNP biogenesis, readily detect the abnormal
pool of mRNAs and degrade them very rapidly in a selective
fashion. Subsequent studies revealed the existence of a number of mRNA quality control pathways working throughout
the entire life of the mRNAs both in cytoplasm as well as in
nucleus, as described later (Fasken and Corbett 2005; Doma
and Parker 2007; Isken and Maquat 2007).
4.
Nature of mRNP aberrations
Aberrant transcripts, in principle, can be generated by two
broad mechanisms. First, a specific mutation within a given
gene may convert the corresponding transcript to an aberrant
state. Second, aberration in a given message may arise in the
absence of a mutation owing to an error-prone transcription
and mRNP biogenesis events in nucleus.
A classic example of first kind of aberration is the transcripts produced from genes containing an in-frame premature termination codon (PTC) due to a mutational alteration
of a genomic sequence either in the ORF or in the upstream
of ORF. Such messages are degraded by one of the mRNA
surveillance systems in the cytoplasm called NMD (see
J. Biosci. 36(3), September 2013
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Satarupa Das and Biswadip Das
below; table 2). Another class of aberrant mRNAs belonging
to this class arises from genomic mutations in genes
encoding any of the splicing and 3′-end forming machinery.
Mutations in these genes generally lead to the splicing and
3′-end formation of corresponding transcripts to take place
either at an inappropriate position or in an improper manner
(see below).
The second mechanism involves an inherent inaccuracy
associated with mRNA processing and biogenesis machinery
in the nucleus that can result in a variety of aberrant and
defective messages even in the absence of any kind of
mutation. Defective splicing or imprecise polyadenylation
in a fraction of a total pool of a given massage can lead to
the formation of defective/aberrant counterpart of that normal message in the cell. Various kinds of aberrations found
associated with mRNAs are described below and are listed in
table 2.
4.1
Aberrant mRNAs resulting from genomic mutations
As mentioned above, this class of defective mRNA is generated as a consequence of genomic mutations in any given
gene that gives rise to following kinds of aberrant messages.
4.1.1 Transcripts containing premature termination codon
(PTC): This is the most well-studied and well-understood
aberrant transcript that harbours an in-frame PTC generated
as result of a mutational insertion within or upstream of ORF
of a given gene (Losson and Lacroute 1979; Leeds et al.
1991; Decker and Parker 1993; He et al 1993; Peltz et al.
1993; He and Jacobson 1995; Czaplinski et al. 1998; Cui et
al. 1999; Hilleren and Parker 1999). Accumulated evidences
have shown that all the PTC mRNAs are targeted for degradation in the cytoplasm by a well-characterized specialized
mRNA decay pathway called nonsense-mediated decay pathway (NMD) (table 2; see below) (Leeds et al. 1991; Decker
and Parker 1993; He et al. 1993; Cui et al. 1999; He and
Jacobson 1995; Czaplinski et al. 1998; Hilleren and Parker
1999). Early work demonstrated that relative location of PTC
in the transcript body has a very important consequence on
the extent of destabilization of PTC-containing mRNA such
that a PTC that is located more towards the 5′-proximity of
the transcript body is more prone to accelerated decay than
the one that harbours the PTC towards its 3′-proximity (Peltz
et al. 1993). To explain this position effect the existence of a
downstream element (DSE) was proposed (Ruiz-Echevarria
and Peltz 1996). Aberrant transcripts harbouring a stop codon
upstream of this cis-acting element were shown to be
destabilized by NMD more efficiently (Ruiz-Echevarria and
Peltz 1996; Ruiz-Echevarría et al. 1998). However, recent
studies discounted the existence of DSE and, instead,
advocated that the abnormal long distance between the
site of translation termination at PTC and the position of
J. Biosci. 38(3), September 2013
the downstream poly(A) tail as demarcated by the
poly(A)-binding protein Pab1p that may generally trigger
the NMD response. This abnormally long distance, which
has been termed a ‘faux’ 3′-UTR (Amrani et al. 2004),
apparently allows the detection of stop codon as premature and subsequent recruitment of the NMD factors by a
poorly understood mechanism (Isken and Maquat 2007)
(see below).
4.1.2 Transcripts that lack natural termination codon: Another
class of aberrant message involves lack of their natural
translational stop codon (Maquat 2002; Frischmeyer et al.
2002; Vasudevan et al. 2002; Wagner and Lykke-Andersen
2002). These can arise when polyadenylation occurs prematurely within a coding region of a gene owing to a mutational event
(Edwalds-Gilbert et al. 1997; Sparks and Dieckmann 1998;
Graber et al. 1999; Frischmeyer et al. 2002) or when the
transcription aborts prematurely (Cui and Denis 2003). It should
be noted here that when a mutational event replaces the natural
stop codon with a meaningful codon, that transcript usually is
not considered by cellular machinery as aberrant because few or
more termination codons naturally always occur at the 3′-UTR
of most messages (Isken and Maquat 2007). Needless to mention that this kind of faulty message will produce defective
polypeptide and is targeted by a specialized quality control
mechanism called non-stop decay (NSD) in cytoplasm (see
below; table 2).
4.1.3 Transcript having a translational elongation stall signal:
These types of defective mRNAs are generated when a
mutational event inserts a non-physiological stem-loop structure
into the transcript body to create a translation elongation stall
signal (Doma and Parker 2007). Once the stem-loop structure is
recognized and a translational elongation pause is detected in
such messages, they are targeted for decay by a specialized
pathway in the cytoplasm called no-go decay (NGD) (Doma
and Parker 2007) (see below; table 2).
4.1.4 Transcripts having mutations in the region of genes those
dictate nuclear mRNA processing events: These kinds of aberrant
messages primarily originate due to cis-acting mutations that take
place either within the vital locations of a gene that takes direct
part in the nuclear mRNA processing event(s) or within specific
signal element(s) that is required for specific mRNA processing
events such as splicing or 3′-end formation. Two of the classic
examples of this kind of mutations involve cyc1-512 (Zaret and
Sherman 1982; Das et al. 2000) and lys2-187 (Chattoo et al.
1979; Das et al. 2006) mutations in yeast Saccharomyces
cerevisiae. The former consisted of a 38 bp deletion at the 3′UTR of CYC1 mRNA that eliminated the critical elements
necessary for 3′-end formation for yeast messages (Zaret and
Sherman 1982; Das et al. 2000) resulting in the formation of
8 aberrantly long cyc1-512 transcripts that were extended to their
Mutation causing alteration
in 3′-end cis-acting signal
sequence,
mRNAs with defect in 3′-end
Formation
(i) Unadenylated
mRNAs with defect in
formation of Export
competent mRNPs
Mutation in genes encoding
3′-end forming machinery
Mutations in genes encoding
mRNA maturing factors
eg. THO/TREX Components
cis-acting mutation in a
specific gene affecting export
competent mRNA formation
Improper Capping
event
Genomic Mutation, Abnormal
inaccurate splicing event
Transcripts having improper
5′-Cap Structure
Splice defective mRNAs
(ii) 3′-extended
Genomic Mutation
Exosome mediated
Decay Pathway in
nulcues, DRN
Exosome Mediated
Decay Pathway in
nucleus, DRN
(i) Nuclear Exosome,
Rrp6p
(i) Exosome mediated
Decay Pathway in
nucleus
(ii) Mlp1p/2p dependent
nuclear arrest
Nuclear Exosome,
Rrp6p, Cbc1p/2p
Nuclear Exosome,
Rrp6p, Cbc1p/2p
(ii) Mlp1p/2p, Pml39p
Hbs1p, Dom34p,
Xrn1p, cytoplasmic
Exosome
Rat1p
Ski2p, Ski3p, Ski7p,
cytoplasmic
Exosome
Upf1p, Upf2p, Upf3p,
Dcp1p/2p, Xrn1p,
cytoplasmic
exosome
Genes/protein factors
No go Decay
(NGD) Pathway
Nonstop Decay
(NSD) Pathway
Genomic mutation causing
internal polyadenylation
within ORF
Transcripts harboring translational
elongation Stall Signal
Nonsense Mediated
Decay (NMD)
pathway
Genomic Mutation
mRNAs with
(i) in Frame PTC,
(ii) long 3′-extended termini
(iii) altered translation initiation,
(iv) uORF
(v) intron containing pre-mRNAs
(vi) mRNAs with frameshifting
mRNAs lacking natural stop codon
Quality control
pathway involved
Cause of aberration
Aberrant mRNP targeted
for quality control
Table 2. Various kinds of aberrations in mRNAs and the quality control pathways
Nucleus
Nucleus
Nucleus
Cytoplasm
Nucleus
Cytoplasm
Cytoplasm
Cytoplasm
Site of decay
Vinciguerra and Stutz (2004),
Jensen et al. (2003)
Zenklusen et al. (2002),
Das et al. (2006),
Kohler and Hurt (2007)
Legrain and Rosbash (1989),
Plumpton et al. (1994),
Bousquet-Antonelli et al.
(2000), Panse et al. (2003),
Hilleren and Parker (2003),
Galy et al.(2004), Sommer
and Nehrbass (2005)
Proweller and Butler (1994),
Burkard and Butler (2000),
Das et al. (2000), Torchet
et al. (2002)
Qu et al. (2009)
Kim et al. (2004)
Edwalds-Gilbert et al. (1997),
Frischmeyer et al. (2002),
Graber et al. (1999), Sparks
and Dieckmann (1998), Van
Hoof et al. (2002)
Doma and Parker (2006)
Losson and Lacroute (1979),
Peltz et al. (1993), Hilleren
and Parker (1999), Cui et al.
(1999), Czaplinski et al.
(1998), Decker and Parker
(1993), Leeds et al. (1991),
Amrani et al. (2004)
Selected references
mRNA quality control pathways in Saccharomyces cerevisiae
621
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622
Satarupa Das and Biswadip Das
3′-termini from 850 bp to more than 3500 bp downstream. These
abnormally long 3′-extended cyc1-512 transcripts were shown to
be preferentially retained in the nucleus (Zaret and Sherman
1982; Das et al. 2000). The latter, lys2-187 mRNA, consisted
of two successive point mutations (one of them is a UGA
mutation) that resulted in the formation of an undesired stemloop structure preventing export of the resulting message from
nucleus to cytoplasm (Das et al. 2006). Both these messages were
targeted for decay in the nucleus by a distinct pathway that is
dependent on nuclear cap binding complex (CBC) called decay
of RNA in nucleus (DRN) (see below; table 2). Similarly, mutations in 5′-splice site and branch point sequence resulted in the
formation of a class of splice defective aberrant messages that are
then exported to the cytoplasm and are degraded there (Legrain
and Rosbash 1989; Bousquet-Antonelli et al. 2000; Hilleren and
Parker 2003).
4.2
Faulty mRNAs resulting from imprecise mRNP
biogenesis
As mentioned above, errors in the mRNA transcripts can
arise even in absence of any mutation via error-prone transcription and inappropriate processing events resulting in the
improper assembly of the mRNP complex in the nucleus.
The following classes of aberrant mRNAs belong to this
category.
4.2.1 mRNAs defective of proper 5′-cap structure: The 5′-cap
structure is characteristic and critical feature of an mRNA.
Capping occurs co-transcriptionally and is one of the earliest
modifications detected on nascent transcripts of 20–30 nucleotides in length (Rasmussen and Lis 1993), soon after its
5′-end emerges from the exit channel of the polymerase
(Shuman 1997). The cap structure has been implicated in many
aspects of mRNA metabolism. It provides resistance to
5′ → 3′ exonucleases (Furuichi et al. 1977; Shimotohno
et al. 1977) and contributes to a variety of cellular processes
including pre-mRNA splicing (Krainer et al. 1984; Konarska
et al. 1984; Edery and Sonenberg 1985; Ohno et al. 1987),
polyadenylation (Cooke and Alwine 1996), mRNA nuclear
export (Hamm and Mattaj 1990; Izaurralde et al. 1992, 1994;
Jarmolowski et al. 1994) and translation (Shatkin 1985).
Experiments from Buratowski lab while addressing the fate
of the mRNA transcripts without a 5′-cap structure, demonstrated that transcription first gets terminated via the ‘torpedo’
model followed by the attack at the unprotected 5′-end by
nuclear exonuclease Rat1p to degrade the nascent uncapped
transcript (Kim et al. 2004). Such direct coupling of the
capping machinery to RNA Polymerase II may ensure that
only those genes that faithfully produce capped RNAs are
properly transcribed and processed further.
J. Biosci. 38(3), September 2013
4.2.2 Splice defective mRNAs: Splicing yields the mature
mRNAs that are devoid of introns. Numerous findings in the
past indicated that the splicing process as a whole is not
absolutely precise. Improper splicing can result in defective
messages that may still retain an intron or lack a given exon
(called skipped exon). In addition, mutation in the splicing
machinery may also result in the generation of varieties of
splice defective aberrant transcripts (consisting of arrested
intermediate at various steps of splicing pathway). A classic
example of this type of aberrant message includes global intron
containing unstable aberrant pre-mRNAs that arise in splice
defective prp2-1 mutant yeast strain (Plumpton et al. 1994).
Splice defective messages were shown both to be retained in
the nucleus as well as to be exported into the cytoplasm
depending on the nature of their defects. For example, mutation in a splicing machinery leading to global splicing defect
(as in case of prp2-1 strain) causes strong retention of global
splice defective intron-containing pre-mRNAs in the nucleus
which are rapidly degraded by nuclear exosome (see below;
table 2) (Bousquet-Antonelli et al. 2000). In addition, the
intron-containing splice defective messages were also demonstrated to anchor at the perinuclear region of the nuclear pore
complex (NPC) by Mlp1p, Pml39p and Nup60p through a
nuclear pathway that is regulated by desumoylating protein
Ulp1p (table 2) (Panse et al. 2003). Anchoring to NPC prevents the nuclear export of these splice defective messages to
the cytoplasm. Yet another class of mutant pre-mRNA in
Saccharomyces, trapped in the nucleus as lariat intermediate
before the second trans-esterification reaction of splicing, was
demonstrated to be debranched, exported to the cytoplasm and
are degraded there (Legrain and Rosbash 1989; BousquetAntonelli et al. 2000; Hilleren and Parker 2003).
4.2.3 Aberrant mRNAs defective in 3′-end formation: Similar
to what was observed in case of splice defective mRNAs,
several mutations in the components of 3′-end formation can
lead to the production of aberrant messages. Such messages
might have a defect either in the cleavage or in the
polyadenylation steps of the 3′-end formation or in both
(table 2). Examples include global 3′-end extended abnormal
transcripts that are generated in rna14-1 and rna15-2 mutant
strains of Saccharomyces cerevisiae (Torchet et al. 2002).
RNA14 and RNA15 genes are the two major components of
yeast cleavage factor CF 1A (Qu et al. 2009; Torchet et al.
2002). The global mRNAs present in rna14-1 and rna15-2
strains have a very long and extended 3′-termini owing to the
defect in cleavage step. These aberrant mRNAs are later
processed by nuclear exosome to yield functional RNAs (see
below; table 2). As mentioned above, eukaryotic RNA
exosome is a large RNA degrading machine that acts on a
variety of RNA substrates in cell including these 3′-extended
aberrant transcripts (section 6.1) (Lykke-Andersen et al. 2011;
Chlebowski et al. 2013). Another class of aberrant mRNA
mRNA quality control pathways in Saccharomyces cerevisiae
belonging to this category is global unadenylatyed messages
generated in pap1-1 mutant strain that is defective in poly(A)
polymerase gene PAP1 and thus cannot polyadenylate the
global mRNAs (Proweller and Butler 1994). These global
unadenylated transcripts are rapidly degraded in Rrp6pnuclear exosome-dependent manner in the nucleus (table 2)
(Burkard and Butler 2000).
4.2.4 Aberrant mRNAs defective in export competent mRNP
formation: The recent body of evidence from a number of
laboratories showed that mRNPs that fail to receive the
correct complements of mRNA-binding proteins are also
regarded as abnormal (Le Hir et al. 2001; Zenklusen et al.
2002; Stutz and Izaurrelde 2003; Jensen et al. 2003;
Vinciguerra and Stutz 2004; Kohler and Hurt 2007; Luna
et al. 2008). As stated above, a number of mRNA maturing
factors collectively called THO and TREX complex remain
associated with growing and maturing mRNP (Proudfoot
et al. 2002; Vinciguerra and Stutz 2004; Kohler and Hurt
2007; Luna et al. 2008). Proper assembly of the maturing
mRNA with the TREX components/export factors to adopt
export-competent mRNP necessitates mRNP export from
nucleus to cytoplasm. Mutation in any of the genes encoding
the components of THO or TREX complex therefore causes
improper assembly of mRNP into an export competent conformation, thus leading to quality control and elimination of
these aberrant messages in nucleus by nuclear exosome (see
below; table 2).
5.
mRNA quality control mechanisms
Given the fact that aberrant mRNAs can be produced in so
many different ways from various sources, it may appear that
eukaryotic cells would be flooded with aberrant and defective messages at any given instance. The current body of
evidence, however, suggests that although aberrant messages
are generated in large abundance, nonproductive polypeptides are seldom produced in the cell, indicating the existence of an elaborate quality control mechanism working
constantly to detect and eliminate the vast majority of aberrant messages. These mechanisms constantly monitor the
various steps of gene expression pipeline for the presence
of such aberrant messages, subsequently detect and sequester
them from the normal cellular messenger RNA pool and
finally destroy them in a preferential manner to prevent their
undesired translation (see below). These quality control
mechanisms work both in the nucleus and cytoplasm as
discussed below.
5.1
mRNA Quality Control in cytoplasm
In Saccharomyces cerevisiae, few mRNA quality control
mechanisms were reported to work in the cytoplasm – NMD,
623
NSD and NGD pathways. Among these the most well-studied
quality control mechanism is NMD pathway (Isken and
Maquat 2007; Rebbapragada and Lykke-Andersen 2009). All
of these systems are described in the following sections.
5.1.1 Nonsense-mediated decay: NMD was discovered in
Saccharomyces as a system that degrades mRNAs with
premature translation termination codon that might have
been incorporated due to a genomic mutation (Losson and
Lacroute 1979). However, the spectrum of aberrant mRNA
substrates that NMD targets are quite wider than just the
class of messages harbouring premature translation codons
(Parker 2012). The list includes (i) mRNAs having long 3′extended termini that alter the proper context of natural stop
codon and poly(A) tail (Muhlrad and Parker 1999; Das et al
2000; Kebaara and Atkin 2009; Deliz-Aguirre et al. 2011),
(ii) mRNAs those have altered translation initiation sites out
of frame of original ORF (Welch and Jacobson, 1999), (iii)
mRNAs with uORF (Gaba et al. 2005), (iv) introncontaining pre-mRNAs with stop codons in the retained
introns (He et al 1993; Sayani et al. 2008) and (v) mRNAs
harbouring frame-shifting where a fraction of translating
ribosomes move out of frame to encounter a stop codon in the
alternative reading frame (Belew et al. 2011) (listed in table 2).
A key feature of NMD is that it requires translation of the
PTC-containing mRNA by ribosomes, and this has been supported by several lines of evidences. First, it has been found
that suppressor tRNA can reduce or even eliminate the degradation of PTC-containing mRNA by NMD (Losson and
Lacroute 1979; Maquat et al. 1981), indicating the strict requirement of translation in NMD. Second, cis-acting mutations
that inhibit the assembly of translation initiation complex
stabilize the PTC-containing messages by blocking NMD
(Belgrader et al. 1994). Third, nonsense-containing transcripts
were found to be polysome associated (Leeds et al. 1991; He
et al. 1993; Stephenson and Maquat 1996; Zhang and Maquat
1997). Finally, drugs that inhibit translation such as cycloheximide can stabilize the PTC mRNAs by abrogating NMD
(Herrick et al 1990; Lim and Maquat 1992; Carter et al.
1995; Zhang and Maquat 1997). Interestingly, in mammalian
cells, NMD targets only the newly synthesized translating
messages that are undergoing the pioneer round of translation
while it is still associated with nuclear cap-binding complex
CBP80/CBP20 (section 1) (Ishigaki et al. 2001). In contrast to
mammalian cells, NMD in Saccharomyces cerevisiae targets
both the newly synthesized (associated with Cbc1p/Cbc2p,
orthologous to mammalian CBP80/CBP20 proteins) as well
as steady state mRNAs (associated with eIF4E) (Gao et al.
2005). It appears that in the first place, NMD targets the
fraction of messages that are bound by nuclear cap binding
complex Cbc1p/Cbc2p followed by targeting the pool of nonsense messages that are bound by eIF4E as well (Gao et al.
2005). This is supported by the finding that S. cerevisiae strain
J. Biosci. 36(3), September 2013
624
Satarupa Das and Biswadip Das
lacking Cbc1p are still viable and is functional for NMD (Das
et al. 2000) by targeting the eIF4E-bound messages (Gao et al.
2005). Second, NMD in S. cerevisiae occurs without significant shortening of poly(A) tail, suggesting that it targets newly
synthesized mRNA (Muhlrad and Parker 1994; Cao and parker 2003). These observations suggest that mRNAs in S.
cerevisiae undergo a CBC1/CBC2-mediated pioneer round of
translation where the PTC-containing transcripts are targeted
for NMD. However, the action of NMD can still continue to
take place in the subsequent steady state rounds of translation
while the mRNAs are bound by eIF4E.
It is believed that during translation every mRNA is monitored for the presence of PTC, and once the translation complex
encounters a termination codon, its context is evaluated by
translational apparatus to verify if the termination codon is
natural or premature (figure 3). If the termination turns out to
be due to PTC, the translation complex disassembles and recruits Upf proteins (originally mistaken as suppressor of frameshift mutation or UP Frameshift) consisting of Upf1p, Upf2p
and Upf3p, which in turn promotes the decapping reaction
mediated by Dcp1p and Dcp2p proteins independent of
deadenylation followed by degradation of transcript body
(Isken and Maquat 2007) (figure 3; table 2). The criterion
by which the context of natural vs PTC codon is evaluated
is not very clear. Current research shows that a long distance between the site of translation termination and the
position of poly(A) tail appears to trigger NMD in
Saccharomyces cerevisiae (Amrani et al. 2004). This abnormally long distance (absent in normal message), which
has been termed as ‘faux’ 3′-UTR, apparently helps
recruiting the NMD factors by a poorly understood mechanism (Amrani et al 2004). In support of this conclusion,
nonsense codon within a given message was shown to
trigger NMD to an extent that depends on its distance
upstream of 3′-UTR (Muhlrad and Parker 1999; Das et al.
2000). Moreover, tethering Pab1p immediately downstream to the site of PTC effectively eliminates faux
3′-UTR function and in turn abolishes NMD (Amrani et al.
2004). This and some other additional data suggests that
normal and natural termination can be distinguished from
abnormal termination by the proximity of the translation termination events to the poly(A)-binding protein Pab1p, which
is independent of the 3′-end cleavage and polyadenylation
(Isken and Maquat 2007).
Once the substrate messages for NMD are identified by the
‘faux’ 3′-UTR in S. cerevisiae as described above, they are
recognized by the concerted action of Upf1p, Upf2p and
Upf3p protein. It has been suggested that in the initial step of
assembly of the NMD factors, Upf1p, a member of SF1 super
family of RNA helicases, interacts with the translation termination complex and alters the nature of translation termination
(Baker and Parker 2004). Upf1p was found to interact with the
other two NMD factors Upf2p and Upf3p as well as the
ribosome release factors eRF1/eRF3. With these and some
J. Biosci. 38(3), September 2013
additional data, it is now believed that translating ribosomes
halt at the premature termination codon followed by the assessment of the context of termination codon. If found premature (as judged by ‘faux’ 3′-UTR), the ribosomes signal the
release factors eRF1-eRF3 as well as Upf1p to be assembled
onto the PTC-containing mRNA. Upf1 then associates with
the nonsense-containing mRNAs as well as with release factors eRF1-eRF3 and subsequently repress the translation of the
nonsense-containing mRNA. In the second step eRF1 dissociates from ribosome after hydrolysis of peptidyl tRNA bond. In
the next step, Upf1p interacts with Upf2p and Upf3p and
promotes their recruitment onto the eRF3-Upf1p-PTC
mRNA complex to form a mature post-termination surveillance complex. Interestingly, binding of Upf2p/3p complex to
Upf1p reduces its interaction with RNA and instead activates
its helicase property (Chakrabarti et al. 2011). This suggests
that after translation termination by Upf1p its interaction with
Upf2p stimulates an alteration of catalytic properties leading to
major mRNP rearrangements by changing the fate of the
translating ribosome. This phenomenon leads to several consequences, e.g. deadenylation of transcript body (Muhlrad and
Parker 1994; Cao and Parker 2003, Mitchell and Tollervey
2003), or rapid deadenylation-independent decapping
(Muhlrad and Parker 1994), enhanced rate of 3′→5′ degradation after deadenylation (Cao and Parker 2003; Mitchell and
Tollervey 2003) and translation repression (Muhlrad and
Parker 1999). Experimental evidence showed that NMD machinery principally targets the nonsense-containing transcripts
in the major decay pathway where the transcript is decapped
independent of deadenylation followed by the degradation of
entire transcript body in 5′→3′ direction by major cytoplasmic
exoribonuclease Xrn1p (Muhlrad and Parker 1994; He and
Jacobson 2001; Cao and Parker 2003; He et al. 2003). In a
second and minor pathway the transcript may also undergo
decay in 3′→5′ direction by exosome-dependent mechanisms
(section 6.1). Recent evidence of accumulation of NMD substrates as translationally repressed RNAs in P-bodies support
the view that translation gets repressed during NMD response,
which is followed by the Upf1p-mediated targeting of the
NMD substrate to these specific cytoplasmic foci (Sheth and
Parker 2006). Finally other Upf factors assemble on these
mRNA substrates and the mRNA undergoes degradation in
these foci following the events described above (Sheth
and Parker 2006; reviewed in Parker 2012). In addition to
aberrant mRNAs, NMD pathway was also demonstrated
to degrade several specific normal mRNAs (Lelivelt and
Culbertson 1999; He and Jacobson 2001; He et al. 2003;
Kebaara and Atkin 2009; Rebbapragada and LykkeAndersen 2009), which is considered as a manifestation
of the regulation of cellular abundance of these messages
by NMD (see below).
5.1.2 Non-stop decay: In addition to NMD, two other specialized
decay pathways, namely NSD and NGD pathways, also operate
in the cytoplasm of S. cerevisiae to selectively destroy two kinds
mRNA quality control pathways in Saccharomyces cerevisiae
625
NUCLEUS
Cbc1P m7G
AAAAAAAA70
UAA
PTC
Export
Shuttling of Released Export Factors
to Nucleus
CYTOPLASM
Mature mRNP in Cytoplasm
Cbc1P m7G
AUG
AAAAAAAA70
UAA
PTC
Pioneer or Steady State Round of Translation
Cbc1P
PTC
Premature Termination
Codon
or m7G
PTC
Upf1p
THO Components
eRF1 eRF3
Export factor Sub2p/
Yra1p/Mex67p
AAAAAAAA70
UAA
Faux 3 -UTR
4E
Nonsense Recognition
Recruitment of Upf1p, eRF1-eRF3
Translation Termination by Upf1p
Cbc1P
Exon Junction Complex
Poly(A) Binding Protein
Pab1p
or
m7G
AUG
Upf1p
Faux 3 -UTR
4E
Ribosome
Cbc1P
4E
Exosome
AAAAAAAA70
UAA
eRF3
Loss of TIFs eg. CBC/4E/4G/eRF1 etc., Translation
Termination, Recruitment of Upf2p/3p and other decay factors
Upf
3p
eRF1
m7G
UAA
AAAAAAAA70
Triggering of mRNA decay via
decapping and 5 3 decay
5 3
Decay
3 5
Decay
Ccr4
Upf1p Upf
3p
Triggering of mRNA decay via
deadenylation and 3 5 decay
PTC
Figure 3. Model of NMD pathway in S. cerevisiae: Newly synthesized messages associated with CBC (undergoing pioneer round of
translation) and steady-state translating mRNAs associated with eIF4E both harboring PTC are targeted by NMD if they have a faux 3′UTR. During the translation of these PTC mRNAs, bound either by Cbc1p/2p or by eIF4E they are subjected to NMD. In the diagram,
relevant mRNA binding proteins which remain associated with PTC mRNAs during different stages of assembly of the NMD components
and decay are shown by colored solid symbols. Each symbol is either annotated directly in the diagram or denoted in the legend box. AUG
and UAA indicate the beginning and end of the open reading frame (ORF) carried by the message. The translating ribosome pauses at the
premature termination codon and signals the release factor eRF1-eRF3 complex so that (1) The NMD factor Upf1p becomes associated with
the eRF1-eRF3 termination complex during the termination process, (2) After hydrolysis of the peptidyl-tRNA bond, eRF1 dissociates from
the ribosome. Dissociation of eRF1 allows Upf2p (and Upf3p) to bind the eRF3-Upf1p complex. (3) Upf2p (or Upf3p) joins the complex
and displaces eRF3 to form the mature post-termination surveillance complex. Subsequently, the aberrant transcript follows either
decapping in deadenylation independent manner followed by degradation of the transcript body in 5′→3′ direction by Xrn1p or
deadenylation dependent mechanism in 3′→5′ by exosome.
of aberrant messages. NSD recognizes aberrant transcripts that
lack any natural stop codon, thus presumably encoding abnormal
polypeptides. Such transcripts arose due to internal and premature
polyadenylation events within a coding region (Edwalds-Gilbert
et al. 1997; Sparks and Dieckmann 1998; Graber et al. 1999;
Frischmeyer et al. 2002). In this process, the ribosome keeps on
translating the aberrant message in absence of the
termination codon until it arrives at the end of the
message. Consequently it then recruits cytoplasmic
exosome and associated Ski protein complex composed
of Ski2p, Ski3p and Ski7p (figure 4A; table 2). The
major player in this game is Ski7p, which is a paralog
of eukaryotic translation elongation factor eEF1A and
eukaryotic release factor eRF3 (Frischmeyer et al.
2002; Van Hoof et al. 2002). The C-terminal domain
of Ski7p resembles the GTPase domain of both of these
proteins (Frischmeyer et al. 2002; Van Hoof et al.
2002). These facts are therefore consistent with a model
where Ski7p recognizes the empty ‘A’ site that is generated when the ribosome arrives at the extreme 3′-end
of the aberrant message and could consequently recruit
cytoplasmic exosome (section 6.1). Notably the NSD
mechanism does not involve decapping and
deadenylation factors, which are typically required for
other pathways of mRNA decay as described above, and
does not require Upf proteins. Interestingly, this
J. Biosci. 36(3), September 2013
626
Satarupa Das and Biswadip Das
b
a
Stable Secondary Structure
Pab1p
mRNA lacking stop codon
m7G
AAAAAAAA70
UAA
Pab1p
m7G
AAAAAAAA70
Ribosome Stalling
Ribosome Stalling
at mRNA 3 -end
m7G
m7G
UAA
AAAAAAAA70
AAAAAAAA70
AUG
Dom
Recruitment
of Ski7p/
Exosome
m7G
AUG
AAAAAAAA70
AUG
m7G
Recruitment
of Dom34p/Hbs1p
AUG
UAA
AAAAAAAA70
Dom
Ski2p
m7G
Recruitment
of Ski2p/3p/8p
Ribosome
Release
Ski7p/
Exosome
AUG
5 Decay by Exosome
5
3 Decay by Xrn1p
Xrn1p
3
Endonucleolytic Cleavage
And Ribosome Release
3
5 Decay by Exosome
Exosome
Figure 4. Model for NSD and NGD in S. cerevisiae: (A) The model for Non Stop Decay envisages that mRNAs lacking a termination
codon are degraded by the exosome independently of deadenylation. The exosome is recruited to the empty A site of the ribosome by Ski7
when the translating ribosome arrives at the end of the extreme 3′-termini, which is a molecular mimic of EF1A and eRF3. (B) The model
for No Go Decay describes that translationally active mRNAs that harbor a strong secondary structure which comes in the way of ribosome
progression are targeted for endonucleolytic decay by a mechanism that requires Dom34 and involves Hbs1. The latter component may be
functionally substituted by Ski7. The resulting 5′ decay product is degraded from the 3′ end by the exosome, and the resulting 3′ Decay
product is degraded from the 5′-end by the Dcp1/Dcp2 decapping complex followed by Xrn1p. For both illustrations, AUG and UAA
indicate the beginning and end of the open reading frame (ORF) carried by the message. Solid blue spheres denote the translating 80S
ribosomes. Only relevant decay components are shown by annotated symbols for simplicity. Proteins which remain associated to
translating/degrading mRNAs during different stages of decay are not shown.
mechanism requires a very special form of exosome that
can degrade the poly(A) tail independently of any
known form of deadenylase complex.
5.1.3 No-go decay: NGD is another quality control mechanism
that targets aberrant mRNAs which harbours a nonphysiological secondary structure causing a strong pause in
the translational elongation (Doma and Parker 2006)
(figure 4B; table 2). NGD requires two essential components,
namely Hbs1p and Dom34p, both of which are paralogs of
translation termination factors eRF3 and eRF1, indicating that
they presumably interact with stalled ribosome. One distinctive
feature of NGD is that it involves one or more endonucleolytic
cleavage in the translating mRNA near the stalled site of ribosome resulting in at least two decay intermediates (figure 4B).
However, currently it is not known with certainty if NGD
involves single endonucleolytic cleavage followed by multiple exonucleolytic decay or multiple endonucleolytic
cleavage. It appears that Dom34p recognizes stalled ribosome by binding to the available ‘A’ site by mimicking
J. Biosci. 38(3), September 2013
a charged tRNA, where together with Hbs1p it may help
recruit the possible endonuclease (figure 4B). Interestingly,
ribosome has also been proposed to cleave the mRNA in E.
coli when it stalls (Tollervey 2006). However, this possibility was never experimentally verified in baker’s yeast.
Notably, a mere pause in the translation elongation does not
activate NGD since the presence of a pseudo-knot structure
or rare codons in the mRNA induce the endonucleolytic
cleavage with much less frequency than an actual stem-loop
structure (Ougland et al. 2004). This indicates that NGD
requires strong pause signal to target and degrade the translating mRNA. Therefore, the pathway appeared to be
evolved to degrade mRNAs that are altered in ways that
cause a complete block in the translation elongation rather
than just slowing down the process.
6.
mRNA quality control in nucleus
Although the nucleus is the site for mRNA biogenesis, this is
the organelle where more than 90% of the cellular RNA
mRNA quality control pathways in Saccharomyces cerevisiae
degradation takes place. The exonucleolytic processing of
ribosomal RNAs (rRNA), small nucleolar RNAs (snoRNA)
and small nuclear RNAs (snRNA) as well as the complete
degradation of spliced introns and internally and externally
transcribed spacers released from ribosomal RNA precursors, all take place in the nucleus. A part of the nuclear
RNA degradation is associated with quality control mechanisms which targets a subset of aberrant mRNAs resulting
from the inaccurate mRNA biogenesis events in the nucleus.
Notably, some kinds of aberrant messages are not exported
efficiently and consequently are retained in the nucleus. In
broad terms, nuclear quality control systems either efficiently degrade these aberrant messages or retain them in a
specified sub-nuclear location to trigger their subsequent
processing. The most well-studied quality control system in
the nucleus is the one mediated by nuclear RNA Exosome
(Mitchell et al. 1997; Allmang et al. 1999). Exosome is a
large RNA degradation machine consisting of ten or more
3′→5′ exoribonuclease which deals with a large share of the
undesired genetic load caused by aberrant mRNAs in the
nucleus (various components of exosome is summarized in
table 3). Another mRNA quality control mechanism operates
in the nucleus that is dependent on nuclear cap binding protein
Cbc1p and nuclear exosome component Rrp6p. This nuclear
pathway targets polyadenylation-deficient cyc1-512 mRNA,
mutant lys2-187 mRNA as well as several normal but special
mRNAs (Das et al. 2000, 2003; Kuai et al. 2005) (see later).
Yet another unique quality control mechanism exists that
checks the competence of the spliced mRNA for export at
the nuclear pore (Sommer and Nehrbass 2005), preventing
the unspliced messages to be exported to the cytoplasm. All
three types of quality control mechanisms are described below.
6.1
Nuclear quality control by exosome
Eukaryotic RNA exosome was discovered in S. cerevisiae
from Tollervey lab as a large molecular machine consisting
of ten or more proteins (of which nine of them are 3′→5′
exoribonucleases) (Mitchell et al. 1997; Allmang et al. 1999;
Butler 2002) involved in the processing of precursors of
ribosomal RNAs into their mature form (Mitchell et al.
1997; Allmang et al. 1999). In subsequent studies, RNA
exosome was found to participate in the degradation of a
wide spectrum of RNA substrates, which include biogenesis
and processing of small nucleolar RNA (snoRNA) and small
nuclear RNA (snRNA) (Allmang et al. 1999; van Hoof et al.
2000). The molecular structure of this large multiprtoein
complex has been deduced from the crystallographic analysis of exosome from Homo sapiens (Liu et al. 2006). The
core of exosome is composed of nine subunits that are
arranged in a two-layered ring structure (Liu et al. 2006;
reviewed in Lykke-Andersen et al. 2011 and Chlebowski
et al. 2013 and the references therein). The bottom layer is
627
composed of six subunits, namely Rrp41p, Rrp42p, Rrp43p,
Rrp45p, Rrp46p, Rrp40p and Mtr3p, into a ‘hexameric’ ring
with a central channel (table 3). All these protein factors
share an overall structural similarity with bacterial phosphorolytic nuclease, RNasePH (reviewed in Lykke-Andersen
et al. 2011 and Chlebowski et al. 2013 and the references
therein). The top layer of the core consists of three proteins
in the shape of a trimeric ‘cap’ complex consisting of
Rrp40p, Rrp4p and Csl4p. Each of Rrp40p and Rrp4p have
one S1 and one KH RNA binding domain and Csl4p contain
one KH domain and a zinc ribbon RNA binding motif
(reviewed in Lykke-Andersen et al. 2011 and Chlebowski
et al. 2013 and the references therein) (table 3). The central
channel formed by the bottom ‘hexameric ring is extended
through the top layer of trimeric cap’. The cap subunits only
make contacts with two subunits of the bottom layer
hexamer and this nine subunit arrangement is known as
Exo-9 (table 3). The central channel that traverses the cap
and hexamer can only accommodate the single-stranded
RNA (Chlebowski et al. 2013).
The Exo-9 subunit is catalytically inactive and is believed
to be critical for the structure of the exosome core and
thought to interact with the RNA substrates. The catalytic
activity is provided by the tenth subunit, Dis3p (a 110 kDa
protein, also known as Rrp44p), which has both endonucleolytic and exonucleolytic activities (Lorentzen et al. 2008;
Schaeffer et al. 2009; Schneider et al. 2009) to form Exo10 (Lykke-Andersen et al. 2011; Chlebowski et al. 2013).
Recent findings suggest that this catalytic subunit is tethered
to Exo-9 ring through the bottom hexamer on the opposite
side of the trimeric cap and is strongly associated with it both
in cytoplasm and nucleus (table 3). In S. cerevisiae, Exo-10
complex in the nucleus also interacts with an additional
3′→5′ exoribonuclease known as Rrp6p (Briggs et al.
1998) to form Exo-11 (Allmang et al. 1999; Liu et al.
2006). Recent electron microscopic analysis of Leishmania
tarentolae exosome suggests that perhaps Rrp6p interacts
with Exo-9 complex towards the vicinity of trimeric cap
opposite to the side where Dis3p interacts with Exo-9, although this is unclear at the moment (Christodero et al.
2008). Sufficiently long RNA substrates may thread through
the central channel formed by the core and cap to arrive at
the active sites of Dis3p/Rrp44p. Alternatively, substrate
RNA may also bypass the core channel and enter the active
site of Dis3p/Rrp44p directly (Wang et al. 2007; Bonneau et
al. 2009). Exosome is able to adapt to a wide spectrum of
RNA substrates and consequently its catalytic activities can
be modulated in various ways based on the nature of the
RNA substrate. This modulation of catalytic activity can be
accomplished by additional protein complexes. Two such
complexes found in the nucleus of S. cerevisiae are known
a TRAMP complex and Nrd1p/Nab3 complex. TRAMP
complex consist of a non-cannonical poly(A) polymerase
J. Biosci. 36(3), September 2013
628
Satarupa Das and Biswadip Das
Table 3. Exosome and exosome-associated proteins involved in quality control of mRNAs
Complex
Structural components and features
Core exosome
(Exo-9)
Six RNasePH domain proteins
forming bottom ring layer Rrp41p,
Rrp42p, Rrp43p, Rrp45p, Rrp46p,
Mtr3p
3 RNA-binding subunits forming cap
or top layer Rrp4p, Rrp40p (Both
contain S1 and KH RNA binding
domain) Csl4p (Contains KH
domain and zinc ribbon RNA
binding motif)
Nuclear and Cytoplasmic Cofactor:
110 kdal Protein, Making contact
with bottom ring layer through
Rrp41p, Member of RNR super
family having both N-terminal
PIN (endonuclease active site)
and C-terminal RNB
(exoribonuclease active site)
domain
Nuclear Cofactor: 84 kdal protein,
3′→5′ exonuriboclease of RNAse
D family, making contact with the
Exo-9/10 with the Cap or top layer.
Dis3p/
Rrp44p
Rrp6p
Rrp47 (Lrp1)
Nuclear Cofactor RNA-binding protein
Mpp6p
Nuclear Cofactor: RNA-binding protein
Ski7p
Binds Csl4 subunit of core exosome and
couples interaction between cytoplasmic
exosome and ski2p/3p/8p complex,
having a eRF3 like GTPase domain
Ski2p: member of ATPase RNA helicase
family
Ski8p:is WD40 repeat protein
Ski3p: TPR protein may function as
scaffold
Contains Mtr4 and a noncanonical
poly(A) polymerase (either Trf4p
or Trf5p)
Ski2p/Ski3p/
Ski8p
complex
Tramp
complexes
Zn-knuckle RNA-binding protein
(Air1p or Air2p)
And RNA helicase Mtr4p
J. Biosci. 38(3), September 2013
Functional characteristics
Selected references
Exo-9: Binding of presentation of RNA
substrates, Scaffolds for interaction with
catalytic subunits and other accessory
proteins
Processing of rRNA
Degradation aberrant rRNAs and mRNAs
in cytoplasm and nucleus
Lykke-Andersen et al.
(2009, 2011), Chlebowski
et al. (2013)
Associated with both cytoplasmic and
nuclear form of exosome ,
Endoribonuclease and 3′→5′
exoribonuclease
Lykke-Andersen et al.
(2009, 2011), Butler and
Mitchell (2011), Chlebowski
et al. (2013)
Associated with only nuclear form of
exosome,
Required for 3′-end processing of 5.8S
rRNA,
Selective decay of aberrant mRNAs in
the nucleus
Involved in retention of several
aberrant mRNAs at sites of transcription
Required for rRNA, snRNA, snoRNA
processing and mRNA surveillance and
degradation,
Associated with nuclear exosome
Required for surveillance of pre-rRNAs
and mRNAs, decay of cryptic Noncoding RNAs
Associated with nuclear exosome
Required for 3′→5′ decay of mRNAs
in cytoplasm, required for non-stop
decay
Lykke-Andersen et al.
(2009, 2011), Butler and
Mitchell (2011), Chlebowski
et al. (2013)
Mitchell et al. (2003), Stead
et al. (2007), Butler and
Mitchell (2011),
Milligan et al. (2008)
Van Hoof et al. (2000, 2002);
Wang et al. (2005), Synowsky
and Heck (2008)
Required for 3′ to 5′ mRNA decay in
cytoplasm
Anderson and Parker (1998);
Brown et al. (2000);Araki
et al. (2001);Wang et al.
(2005), Synowsky and
Heck (2008)
Polyadenylates RNA substrates e.g. aberrant
noncoding RNAs generated by pervasive
Pol II transcription, biogenesis and
turnover of functional coding and
noncoding RNAs
Stimulates processing/degradation of
RNA substrates in poly(A)-dependent
and independent manners by recruiting
exosome to substrates
Houseley and Tollervey
(2006); San Paolo et al.
(2009); Butler
and Mitchell (2011),
Schmidt and Butler
(2013)
mRNA quality control pathways in Saccharomyces cerevisiae
Trf4p or Trf5p, a zinc knuckle protein Air1p or Air2p, and
the RNA helicase Mtr4p (Lacava et al. 2005; Vanacova et al.
2005; Wyers et al. 2005; Houseley et al. 2006). Nrd1p/
Nab3p heterodimer complex (Vasiljeva and Buratowski
2006) is involved in transcription termination of RNA polymerase II transcripts (Steinmetz and Brow 1998; Conrad
et al. 2000; Steinmetz et al. 2001). Interestingly, TRAMP
complex (Trf4p/5p/Air1p/ 2p/Mtr4p) or Nrd1p/Nab3p complex is found associated with Exo-11 (Exo-9+Dis3p+Rrp6p)
complex only in the nucleus of Saccharomyces. On the
contrary, Exo-10 (Exo-9+Dis3p) in cytoplasm has been
demonstrated to be associated with additional cytoplasm
specific co-factors Ski2p/3p/8p (Lykke-Andersen et al.
2009; Klauer and van Hoof 2013). The existence of distinct
nuclear and cytoplasmic forms of exosome indicates that
exosome exists in multitudes of functional forms acting on
discrete and specific sets of RNA substrates. This substrate
specificity of various forms of RNA exosome arises due to
association of core exosome with various co-factors in different sub cellular locations which may also distinguish
between the various modes of actions (e.g. processing vs
degradation) of exosome under various circumstances. In
addition, nuclear form of exosome is also associated with
two other accessory factors known as Lrp1p or Rrp47p
(specifically associates with Rrp6p) and Mpp6p (table 3)
both of which are necessary for function of nuclear form of
exosome (Mitchell et al. 2003; Milligan et al. 2008; Butler
and Mitchell 2011). While cytoplasmic exosome is involved
both in the general and default decay of all mRNAs as well
as in the selective decay of aberrant mRNAs in the cytoplasm (discussed above in the previous sections), nuclear
exosome is involved in the accelerated decay and processing
events of non-coding RNA precursors and surveillance and
selective decay of varieties of pre-mRNAs and mRNAs in
nucleus (discussed below in this section).
The exosome was found to degrade virtually all classes of
RNA substrates including both the coding and non-coding
class of RNAs such as rRNAs, snRNAs, snoRNAs, pretRNAs, pre-mRNAs and mRNAs. Functionally, the nuclear
form of exosome is involved in maturation/ trimming/processing of various ribosomal RNAs and sn/snoRNA precursors,
degradation of unstable RNAs generated by pervasive and
read-through transcription, elimination of excised introns and
other byproducts of gene expression. It also plays a functional
role in the regulated decay of pervasive transcripts generated
by RNA polymerase II all over the yeast genome including,
cryptic unspliced transcripts (CUTs), stable un-annotated transcripts (SUTs) and short promoter-associated RNAs (PARs)
(Davis and Ares 2006; Neil et al. 2009; Wyers et al. 2005; Xu
et al. 2009). Recent transcriptome-wide analyses of different
classes of substrate RNAs and their mapping and interaction
with individual exosome subunits very clearly defined the
nature, abundance and multitude of exosome substrates and
629
the relative contribution of catalytic subunits Dis3p and Rrp6p
in their degradation (Gudipati et al. 2012; Schneider et al.
2012). These studies revealed that (i) a large amount pretRNAs, unspliced pre-mRNAs and some pol III transcripts
are extensively degraded by exosome (Gudipati et al. 2012;
Schneider et al. 2012), (ii) majority of RNAs produced in wildtype cells are eliminated by exosome before entering into
functional pathways (Gudipati et al. 2012) and (iii) all classes
of RNAs were first oligoadenylated followed by degradation
by exosome (Schneider et al. 2012). Dis3p and Rrp6p – the
two catalytic subunits of exosome – have both distinct as well
as overlapping functions in degrading specific sets of substrates (Gudipati et al. 2012) and they appear to co-operate
and communicate with each other to catalyze the decay of their
overlapping substrates (Gudipati et al. 2012; Schneider et al.
2012).
In addition to its role in the biogenesis/processing/surveillance of non-coding RNAs, the exosome was also implicated
in the degradation of a variety of normal and abnormal/
aberrant mRNAs both in the cytoplasm and nucleus in S.
cerevisiae. In the nucleus, this molecular machine has been
reported to degrade nuclear pre-mRNAs (BousquetAntonelli et al. 2000), unadenylated (Burkard and Butler
2000) and 3′-extended unprocessed pre-mRNAs (Torchet
et al. 2002) as well as mRNP assembled in yeast strains in
which components of the TREX (transcription-export) (see
above) complex are dysfunctional (Libri et al 2002;
Zenklusen et al. 2002; Jensen et al. 2003). The first evidence
for a nuclear quality control function by exosome came from
observation that depletion of 3′→5′ exoribonuclease, Rrp6p,
a component of nuclear exosome could suppress the
polyadenylation defect of pap1-1 mutation (Burkard and
Butler 2000). Pap1p protein encodes the major poly(A)
polymerase enzyme, which is involved in coupled
polyadenylation of 3′-end cleavage reaction (MinvielleSebastia and Keller 1999). This study revealed that the
global unadenylated mRNAs generated in pap1-1 mutants
are rapidly degraded by Rrp6p as a part of nuclear quality
control of aberrant mRNAs (Burkard and Butler 2000).
RNA exosome also selectively degrades the introncontaining pre-mRNAs that accumulate in a temperaturesensitive splicing defective prp2-1 strain (Bousquet-Antonelli
et al. 2000). Prp2p is a protein of DEAH box family of ATP
dependent RNA helicase that is required prior to the first transesterification reaction and is released from spliceosome following ATP hydrolysis (Plumpton et al. 1994). This protein is
not essential for spliceosome assembly and the spliceosome
remains intact and associated with pre-mRNA in prp2-1 strain.
The intron-containing splice defective pre-mRNAs accumulate to approximately 20- to 50-fold in a exosome defective
prp2-1 GAL:rrrp41 strain (Rrp41p depleted) compared to a
prp2-1 strain, where the exosome remained functional
(Bousquet-Antonelli et al. 2000). However, the levels of their
J. Biosci. 36(3), September 2013
630
Satarupa Das and Biswadip Das
corresponding mature mRNAs or those of the other mRNAs
lacking introns remained unaffected in those strains
(Bousquet-Antonelli et al. 2000). These results indicated that
nuclear exosome is involved in the selective and rapid elimination of various nuclear pre-mRNAs in order to ensure that
these pre-mRNAs are not exported and subsequently translated. The rapid decay of these nuclear pre-mRNAs requires the
activity of core exosome component such as Rrp41p, nuclear
exosome component Rrp6p, a 3′→5′ exoribonuclease as well
as Rat1p, a nuclear 5′→3′ exoribonuclease. Experiments addressing the directionality of the decay mechanism and the
relative contribution indicated that 3′→5′ decay component
has a higher contribution, forming a major pathway of decay.
These findings were also corroborated by the recent
transcriptome-wide analyses where large amount of various
intron-containing nuclear pre-mRNAs were found crosslinked to Rrp44p/Dis3p subunit indicating that Rrp44p is
involved in the degradation of these unspliced or partially
spliced RNA species. Moreover, Rrp44p sequence coverage
over the long intron-containing pre-mRNAs is much higher
than those of short inron-containing pre-mRNAs (Schneider et
al. 2012), which is also consistent from the studies by Gudipati
et al. (2012), who demonstrated that pre-mRNAs are preferentially degraded by the catalytic subunit Rrp44p. Finally
these studies also suggested that a competition appear to exist
between splicing and nuclear decay activity and that the decay
pathway was subjected to metabolic regulation by glucose
(Bousquet-Antonelli et al. 2000; Gudipati et al. 2012).
Evidence from Tollervey lab also indicated that the
exosome functions to process 3′-extended read-through transcripts in rna14-1 and rna15-2 strains to generate functional
mRNAs (Torchet et al. 2002). In S. cerevisiae as well as in
other eukaryotes, the mature 3′-ends are generated by an
extensive processing event that involves a site-specific
cleavage towards the 3′-end of the transcript and the subsequent polyadenylation which are functionally coupled to
transcription termination (Minvielle-Sebastia and Keller
1999). RNA14 and RNA15 genes are the two critical components of this 3′-end maturation event and both of them are
required for this cleavage and coupled polyadenylation
(Minvielle-Sebastia et al. 1991, 1994). The poly(A) polymerase is also a component of the system that is involved in
the polyadenylation reaction. Several mutations such as
rna14-1 and rna15-2 inhibit this reaction, thus resulting in
the defective long 3′-extended read-through transcripts that
are very unstable (Birse et al. 1998; Yonaha and Proudfoot
2000; Minvielle-Sebastia and Keller 1999; Proudfoot 2000).
These transcripts are greatly stabilized in strains depleted in
core exosome component Rrp41p and RNA helicase Dob1p/
Mtr4p, indicating that these long aberrant transcripts undergo degradation by core exosome in a Dob1p-dependent
manner. Interestingly, when Rrp6p, the nuclear exosome
component was depleted in rna14-1 background- it gave a
J. Biosci. 38(3), September 2013
very different phenotype. Short polyadenylated species of
translationally competent pre-mRNAs were found to accumulate greatly in this background, which were destabilized
when Rrp41p, the major core exosome component, was depleted. These findings suggested that exosome processively
degrade these aberrant 3′-extended read-through transcripts
via 3′→5′ direction till exosome arrives at a site close to the
polyadenylation site. In galactcose medium, these intermediates undergo an uncoupled polyadenylation in an rna14-1
rrp6-Δ background to achieve their functional form, while in
glucose medium those transcripts are completely degraded by
exosome (Torchet et al. 2002). Data from these set of experiments indicate clearly that the exosome plays a critical role in
the nuclear surveillance of 3′-extended messages that are generated in cleavage/polyadenylation defective rna14-1 and
rna15-2 mutant strains (Torchet et al. 2002).
In strains having mutation in the THO complex, which is
involved in the mRNP assembly as well as transcription
elongation, several mRNAs fail to receive the appropriate
complement of proteins and are thus unable to assemble into
an export competent mRNP. These aberrant mRNPs are
rapidly degraded by a mechanism that requires the catalytically active Rrp6p and TRAMP complex poly(A) polymerase Trf4p (Libri et al. 2002; Rougemaille et al. 2007;
Assenholt et al. 2008). Notably, the degradation of these
mRNAs still continues even in presence of an altered variant
of Trf4p protein that is defective in polyadenylation, indicating that Trf4p can recruit the exosome in a polyadenylationindependent manner (Rougemaille et al. 2007). Finally the
discovery that THO components genetically interact with 3′end processing factors and the finding that THO mutants are
defective in polyadenylation suggests that defects in proper
mRNP assembly may lead to improperly polyadenylated
mRNAs having much shorter poly(A) tails. Such mRNAs
are recognized and degraded by Rrp6p and possibly by
nuclear exosome (Saguez et al. 2008).
In addition, function of the exosome or at least Rrp6p
action is required for the accumulation of some kinds of
aberrant transcripts at or near the transcription site in the
nuclear foci. In S. cerevisiae, mutant strains defective in
THO complex component sub2-1, global mRNAs fail to
assemble into functionally export-competent mRNPs. Heat
shock HSP104 mRNA in this strain, which presumably fail
to assemble into proper export competent form, were shown
to be retained near their transcription associated foci in an
Rrp6p-dependent manner (Jensen et al. 2001; Libri et al.
2002). Moreover, unadenylated and hyperadenylated versions
of HSP104 mRNAs those are generated in polyadenylation
deficient pap1-1 yeast strain and in export defective rat7-1 or
rip1-Δ yeast strains respectively are also retained close to the
site of transcription, which is dependent on Rrp6p (Hilleren et
al 2001). Interestingly, synthetic reporter mRNA lacking
poly(A) tail were reported to retain near transcription sites,
whereas reporter transcript with a DNA encoded poly(A) tail
mRNA quality control pathways in Saccharomyces cerevisiae
was released from such sites. This implies that the existence of
poly(A) tail is critical for the release from transcription site
(Dower et al. 2004). Furthermore, all these data indicate that
Rrp6p and perhaps the exosome may tether these aberrant
messages to the site of transcription via RNA polymerase II
and chromatin. The molecular mechanism of exosomedependent transcription site retention of aberrant transcript is
unclear at this time. It is currently perceived that retention of
aberrant transcripts could allow these defective mRNAs to be
further processed or could prevent additional errors in mRNP
production from taking place. Taken together, it is now well
accepted that nuclear exosome and Rrp6p is responsible for
rapid elimination of a wide variety of aberrant and defective
mRNAs and retention of some defective mRNAs to the site of
transcription.
6.2
Nuclear quality control by DRN
Besides the exosome-dependent nuclear surveillance pathways, another novel pathway of nuclear mRNA decay has
also been described, which was demonstrated to be distinct from the default and regulated cytoplasmic mRNA
decay and quality control pathways. This mechanism is
dependent on the nuclear cap binding complex CBC and
nuclear exosome component Rrp6p (Das et al. 2000;
2003). This mRNA decay/quality control system was
named DRN (decay of RNA in the nucleus) and it is
now perceived that DRN is a nuclear pathway of quality
control mechanism of messenger RNA in S. cerevisiae
which degrades some specific classes of aberrant mRNAs
(figure 5; table 2). This pathway was also shown to
selectively degrade a small subset of normal messages
called special messages (see below).
DRN was uncovered during the investigation of the
mechanism of suppression of a unique mutation in CYC1
gene encoding the iso-1-cytochrome c protein. As mentioned
before, this mutation, cyc1-512, is a cis-acting 3′-end
forming defect that resulted in the formation of eight aberrantly long 3′-extended transcripts (Zaret and Sherman 1982;
Das et al. 2000) that were unstable and were shown to be
degraded very rapidly, thus causing a 90% reduction in the
level of cyc1-512 mRNA (Das et al. 2000). The existence of
DRN was proposed from the finding that mutation and/or
deletion of CBC1 and RRP6 gene could suppress the deleterious effect of cyc1-512 mutation (Das et al. 2000; 2003).
Further studies revealed that Cbc1p and Rrp6p were responsible for the rapid nuclear decay of these aberrant and unstable long 3′-extended cyc1-512 transcripts.
The Cbc1p-dependent DRN system was postulated as a
nuclear pathway from the experiments where it was demonstrated that normal and global cellular mRNAs were retained
in the nucleus of temperature-sensitive mRNA export defective mutant yeast strain nup116-Δ (Wente and Blobel 1993)
631
at the non-permissive temperature of 37°C (when nuclear
export ceases) became dramatically destabilized (Das et al.
2003). This finding indicated that global mRNAs under this
condition of nuclear retention undergo rapid degradation
(Das et al. 2003). Deletion of either of CBC1 or RRP6 under
the same condition resulted in diminished decay and concomitant stabilization of all of these transcripts, suggesting
that this accelerated decay is dependent on Cbc1p and Rrp6p
(Das et al. 2003). These findings established the existence of
DRN system as a distinct decay mechanism that functions in
the nucleus and also suggested that both Cbc1p and Rrp6p
are required for such decay of normal global cellular transcripts. Consistent with this view, rrp6-Δ was also found to
suppress cyc1-512 mutation and CBC1 and RRP6 gene were
found to constitute a genetic epistatic group for this nuclear
degradation (Das et al. 2003). DRN system also requires the
nuclear 5′→3′ exoribonuclease Rat1p, which constitutes a
minor pathway while the major mechanism of this system
involved Rrp6p mediated 3′→5′ degradation. Based on these evidences, Das et al. (2003) suggested that this novel
nuclear mRNA decay pathway (DRN) operates on all
mRNAs, where the extent of stabilization/degradation of a
given mRNA depends on the degree of its nuclear export/
nuclear retention (Das et al. 2003). Since some aberrant
mRNAs tend to retain completely in the nucleus due to
inefficient 3′-end processing (Eckner et al. 1991; Huang
and Carmichael 1996) or due to some other structural aberrations, e.g. cyc1-512/lys2-187 mRNAs (Das et al. 2000; 2006),
DRN was proposed to act very specifically and selectively on
these mRNAs in the nucleus and postulated to function as a
quality control mechanism to limit their unnecessary and
unwanted accumulation. In case of a normal strain, appropriately processed mRNPs are exported very efficiently from the
nucleus to cytoplasm and hence could escape the DRN action.
Consistent with this view, DRN system was demonstrated to
act on another mutant mRNA known as lys2-187 mRNA (Das
et al. 2006), while the normal LYS2 or other general pool of
normal mRNAs were not degraded by DRN (Das et al. 2006).
LYS2 gene in yeast encodes an important enzyme in the lysine
biosynthesis pathway known as α-aminoadipate reductase
(Chattoo et al. 1979). This mutation caused a structural defect
in the appropriate export-competent conformation of the mutant lys2-187 message, thus prompting the mutant message to
be retained in the nucleus (M Salim, SV Harding, M Seetin,
DH Mathews and F Sherman, personal communication).
Notably, these studies also revealed the existence of a
kinetic competitive mechanism that operates between two
mutually competitive processes such as nuclear export and
nuclear degradation (Das et al. 2003). Various kinds of
aberrant mRNAs, as mentioned above, were shown to be
retained in the nucleus to various extent either due to cisacting defects such as in the case of cyc1-512 or lys2-187
mRNAs or due to trans-acting defect such as global export
defect in nup116-Δ strain and thus became extremely
J. Biosci. 36(3), September 2013
632
Satarupa Das and Biswadip Das
DNA
Nuclear Degradation
Retention at Specific Nuclear Foci
Transcription
Pre-mRNA
UAA
D
e
f
e
c
t
i
v
e
capping
Capped mRNA Cbc1P m7Gppp
UAA
Splicing
Spliced mRNA
Cbc1P m7Gppp
UAA
UAA
DRN/Rrp6p
Recruitment of
Exosome or DRN
components
Cbc1P
AUG
AAAAAAAA70
P
r
o
c
e
s
s
i
n
g
UAA
AAAAAAA
Cbc2P
Cbc1P
Polyadenylation
Polyadenylated
and Export Cbc1P m7Gppp
Competent mRNA
Exosome
OR
UAA
Cbc2P
Cbc1P
UAA
AAAAAAA
Cbc2P
Aberrant mRNAs
Export
NUCLEUS
Shuttling of Released
Export Factors to Nucleus
THO Components
Export factor Sub2p/
Yra1p/Mex67p
CYTOPLASM
Cbc1P
Exon Junction Complex
Poly(A) Binding Protein
Pab1p
or
m7Gppp
AAAAAAAA70
Mature Translating mRNA
4E
Ribosome
Translation
Degradation
Figure 5. Model of nuclear surveillance pathway in S. cerevisiae: Schematic diagram showing the stages of mRNA biogenesis in nucleus
each of which is subject to quality control by nuclear surveillance systems such as nuclear exosome or DRN. As shown in the figure bulk
normal mRNAs after synthesis and processing are exported quickly out of the nucleus into the cytoplasm. Aberrant messages on the other
hand are not exported rapidly and spend longer time in the nucleus and subjected to the action of exosome and DRN. The coupling of
various biogenesis events to quality control systems are represented by dashed arrow. Aberrant messages are thus quickly destroyed by
exonucleolytic action of either exosome/DRN which maintains the fidelity of gene expression. Various mRNA binding proteins which are
deposited onto/remain associated with maturing transcripts during different stages of nuclear phase of life are shown by colored solid
symbols on the transcript body. Each symbol is either annotated directly in the diagram or denoted in the legend box. The release of the
THO component/ maturing factors are shown in appropriate places. The factors/components shown in green, yellow or magenta ellipsoids
in the nuclear decay complex are hypothetical whose identity is still unknown. AUG and UAA indicate the beginning and end of the open
reading frame (ORF) carried by the message. The association of the translating mRNAs with either CBC or eIF4E are shown as before.
sensitive to DRN. This long retention time of the aberrant
mRNPs in the nucleus, however, allows the possibility that
aberrant mRNPs have another opportunity to rectify the
defect associated with them (Doma and Parker 2007).
6.3
Nuclear quality control at the nuclear pore
In the recent past, another novel and interesting kind of
quality control mechanism was reported which works at the
nuclear pore complex (Sommer and Nehrbass 2005). As
mentioned above, once the mRNA transcripts are synthesized, processed and become matured in the nucleus, they
first achieve a proper export-competent conformation
(Jensen et al. 2003; Stutz and Izaurrelde 2003) before they
J. Biosci. 38(3), September 2013
can be exported from nucleus to cytoplasm (Dimaano and
Ullman 2004). One aspect of their maturation event involves
the binding and assembly of these transcripts with the export
receptor which targets these assembled mRNPs to the NPCs
for translocation to cytoplasm (Suntharalingam and Wente
2003). It is now recognized that the assembly, maturation
and accomplishments of export-competence are intimately
coupled to the quality control of the exporting mRNPs. This
quality control mechanism checks the export (in)competence
(such as alterations of coding sequence/absence of mRNP
maturing factors/lack of specific processing events) of the
mRNPs when they arrive at the nuclear pore complex. Here,
the defective mRNP is subject to quality control, which
triggers their selective destruction. Global transcripts not
associated with splicing and export factors sub2p and
mRNA quality control pathways in Saccharomyces cerevisiae
Yra1p respectively in THO mutant strain is the example of
this kind of export-incompetent messages that undergo rapid
elimination by nuclear exosome (Zenklusen et al. 2002).
In addition to the degradation of inappropriately assembled mRNPs, translocation of such mRNPs from nucleus to
cytoplasm is also scrutinized, as evidenced by several recent
studies which demonstrated that at least two myosin-like
proteins in budding yeast known as Mlp1p (Galy et al.
2004) and Mlp2p (Strambio-de-Castillia et al. 1999) assure
that only mature and fully processed mRNPs are exported to
cytoplasm (Sommer and Nehrbass 2005). These Mlp proteins are localized in perinuclear locations at the inner basket
of the NPC and are believed to make contact with exporting
mRNPs when they are targeted for translocation. They were
first implicated in this type of quality control from studies in
which it was demonstrated that their over expression led to
the nuclear accumulation of some mRNAs (Bangs et al.
1998; Kosova et al. 2000) and they interact directly with
yeast hnRNP Nab2p (Green et al. 2003). More direct demonstration of the involvement of these proteins came from
Nehrbass lab, where it was demonstrated that (i) mutation in
Mlp1p allow unspliced messages to leak from nucleus to
cytoplasm and (ii) Mlp1p also interact with splice-site binding protein SF1 and branch-point-binding protein (BBP) in a
RNA-dependent manner (Galy et al. 2004). These observations led to a model in which it was recognized that mRNPs
right before export were retained and subjected to a final
round of quality control until the complete processing is
done (Galy et al. 2004).
Interestingly, there is a major difference in mechanism
between various nuclear quality control of mRNP biogenesis. The quality control mechanism achieved both by nuclear
exosome and DRN is associated with specific nuclease activity, whereas the other kind of control mediated by Rrp6p
and Mlp1p/2p apparently does not require any such degradation mechanism. Although the existence of Mlp-triggered
ribonuclease activity was postulated, it is not yet identified
(Fasken and Corbett 2005). Current evidence strongly suggests that Mlp proteins act like faithful gatekeepers. They
issue a gate pass to the passenger mRNAs to be exported
after careful examination, which impact their movement
across the nuclear pore without destroying them (Sommer
and Nehrbass 2005).
7.
A direct role for mRNA degradation pathways
in the regulation of gene expression?
In recent years, a large body of evidence demonstrated
directly or indirectly that regulation of gene expression can
be achieved at both transcriptional and post-transcriptional
level where differential degradation of mRNAs may play a
crucial and direct role (Caponigro and Parker 1996;
McCarthy 1998; Kuai et al. 2005; Tran and Wente 2006;
633
Isken and Maquat 2007). It now appears that, although
quality control pathways originally had evolved as a mechanism to scrutinize the accuracy of the biogenesis of mRNA,
they also play an important functional role in the control of
gene expression at least in Saccharomyces. Several recent
studies uncovered that surveillance and quality control pathways are also involved in degradation of some otherwise
normal transcripts in the cell. These findings hint that specialized mRNA decay pathways may be engaged in the
control of the physiological steady state level of mRNA
abundance in the cell that is accomplished at the posttranscriptional level by selectively degrading a specific message in response to specific environmental cue.
The first clue that quality control pathways may play roles
in controlling the normal physiological repertoire of messenger
RNAs came from Culbertson’s lab (Lelivelt and Culbertson
1999). They demonstrated that while the abundance of majority of mRNAs remained unaffected by inactivation of NMD
pathway in S. cerevisiae, a small group of 225 normal messages were found to increase in their abundance and stability
from 2- to 11-fold in the upf strains (Lelivelt and Culbertson
1999). However, the up-regulation/stabilization of these normal messages were found to be due to a secondary consequence of stabilizing effect of mRNAs encoding various
transcription regulators in upf background (Lelivelt and
Culbertson 1999).
However, it was not clear why these mRNAs encoding these
transcription factors were targeted by NMD in the absence of
any premature nonsense codons. Although an alternative mechanism was proposed to explain the susceptibility of these
mRNAs to NMD, no clear-cut evidence of such mechanism
was presented. Consistent with this view, many mRNAs were
shown to increase or decrease without altering their half-life in
upf strains. Other genetic and genome-wide analyses in yeast
strains defective in NMD factors and general decay factors such
as upf1-Δ, upf2-Δ, upf3-Δ, dcp1−Δ and xrn1-Δ strains revealed that NMD factors are involved in the regulation of
approximately 765 transcripts in the total yeast cellular
mRNAome that comprises the core substrates of the NMD
pathway (He and Jacobson 2001; He et al. 2003). Subsequent
classification of these transcripts revealed that these ‘normal
core substrates’ include (1) mRNAs with +1 ribosomal
frameshifting, (2) mRNAs encoded by pseudogenes, (3)
bicistronic mRNAs and (4) transcripts encoded by transposable
elements (He et al. 2003) other than the previously known
studied class of substrates as described above. However, no
mechanistic insight about specific feature(s) responsible for the
susceptibility of these normal mRNAs to NMD pathway was
uncovered.
Recent studies have shown that indeed NMD contributes
to the post-transcriptional regulation of gene expression in
both yeast and human cells as evidenced by the consequent
enhancement of a number of endogenous mRNAs lacking
any premature termination codon in cells depleted with
J. Biosci. 36(3), September 2013
634
Satarupa Das and Biswadip Das
NMD factors (Kebaara and Atkin 2009; Yepiskoposyan et
al. 2011). While analysing the specific feature of these
endogenous mRNAs targeted by NMD, these authors provided evidence that mRNAs that were found to be upregulated by NMD abrogation had a greater median 3′UTR length compared to that of the general mRNAome
(Kebaara and Atkin 2009;Yepiskoposyan et al. 2011).
Some of the endogenous substrates were also found to be
enriched in 3′-UTR introns and uORFs (Yepiskoposyan et al.
2011). More interestingly, the mRNAs encoding the NMD
factors themselves also have longer than average 3′-UTR, thus
posing an exciting possibility that these messages could be the
substrates of NMD and are auto-regulated by their own protein
product. Since long 3′-UTR (such as faux 3′-UTR, as stated
above) in general was found to be the critical feature that
targets a given message to NMD machinery, it can be
ascertained that this feature is a frequent trigger for NMD
which is utilized by eukaryotic cells to regulate a small subset
of their genes at the post-transcriptional level by NMD
(Yepiskoposyan et al. 2011).
DRN pathway also targets nucleus-arrested global normal mRNAs accumulated in the nucleus due to defects
associated with mRNA processing (e.g. 3′-end forming
defect) and/or export (e.g. nup116-Δ defect) (Das et al.
2000, 2003). These experiments indicated that DRN could
potentially target normal mRNAs if they are retained in
the nucleus. Based on this assumption, DRN was demonstrated to act on a small subset of 25–30 normal transcripts (referred to as special mRNAs) even in the
absence of any kind of defect in export or processing
(Kuai et al. 2005). Subsequent studies established that
DRN preferentially degrades them in order to control
their cellular and physiological abundance (Kuai et al.
2005). These mRNAs are selectively targeted because
their export is very slow compared to bulk normal and
typical mRNAs. Preliminary evidence has demonstrated
that a special mRNA, HAC1 lacking any notable aberration, undergoes rapid DRN-dependent degradation (Sarkar
and Das, unpublished observation). Interestingly, Hac1p is
bZIP class of transcription factor (Ron and Walter 2007)
that triggers the induction of the important intracellular
signalling pathway called Unfolded Protein Response
(UPR) in response to endoplasmic reticular stress
(Merksamer and Papa 2010, Matus et al. 2011; Back
and Kaufman 2012.). Notably, UPR was implicated in a
variety of neurological and metabolic disorders such as
Alzheimer’s and Parkinson’s diseases and diabetes (Back
and Kaufman 2012). Although it is premature at this
point of time to conclude strongly, current evidences
indicate that preferential degradation of these special messages by DRN in the nucleus represents a novel mode of
post-transcriptional regulation of expression of genes. This
regulation presumably involves preferential modulation of
J. Biosci. 38(3), September 2013
nuclear export and/or decay of these special messages by
DRN. However, the specific feature(s) attributable to slow
export of these mRNAs is currently not known. Although
it has been postulated that special mRNAs harbour a cisacting export retarding element which may slow down the
export of these messages, existence of such element has
yet to be demonstrated experimentally. Efforts are currently under way to dissect out the existence and nature
of such element(s) responsible for slow export of this
special class of mRNAs which appears to be the major
underlying principle of this unique gene regulation (Bugh
and Das, unpublished observation).
8.
Future research avenues
Although the phenomenon of mRNA quality control was
discovered in Saccharomyces cerevisiae, some of these pathways also exist in metazoans since the aberrant and abnormal
mRNAs are also known to be generated in higher mammals
and humans. It is very evident that the amount and diversity of
aberrant messages in metazoans and humans are expected to
be much higher. Since aberrant messages are known to be
associated with a number of diseases, they pose a big threat
to humans. Moreover, the mechanism to distinguish the normal messages from the abnormal pool is very complicated in
higher mammals and is quite different from yeast is a subject
of active research. Future research should be targeted to identify the complete set of genes involved in the quality control of
mammals and its mechanistic details, which in turn should
reveal the molecular link between the mRNA quality control
and some of the human genetic diseases.
As mentioned above, a small group of genes in yeast
Saccharomyces cerevisiae were demonstrated to be regulated
in unique fashion by DRN (Kuai et al. 2005). Although this
observation revealed a new modus operandi of eukaryotic
gene expression, its underlying mechanism is still unknown.
Understanding the mechanisms involved in this new paradigm
of gene regulation is an open and intriguing question. New
research effort is therefore imperative to dissect out the molecular nature of the control of their expression by DRN.
Acknowledgements
Research work at author’s laboratory is funded by research
grant from Council of Scientific and Industrial Research
(Ref. No 38/1280/11/EMR-II), Department of Science and
Technology (File No. SR/SO/BB/0066/2012) and a Research Grant from Jadavpur University to BD and from
Department of Science and Technology, India, to SD (SR/
WOS-A/LS-258/2010).
mRNA quality control pathways in Saccharomyces cerevisiae
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MS received 12 December 2012; accepted 02 May 2013
Corresponding editor: SUDHA BHATTACHARYA
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