Journal of Experimental Botany, Vol. 63, No. 4, pp. 1663–1673, 2012
doi:10.1093/jxb/err401 Advance Access publication 3 December, 2011
REVIEW PAPER
The cutting crew – ribonucleases are key players in the
control of plastid gene expression
Rhea Stoppel and Jörg Meurer*
Biozentrum der Ludwig-Maximilians-Universität, Plant Molecular Biology/Botany, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany
* To whom correspondence should be addressed. E-mail: [email protected]
Received 15 September 2011; Revised 28 October 2011; Accepted 16 November 2011
Abstract
Chloroplast biogenesis requires constant adjustment of RNA homeostasis under conditions of on-going developmental and environmental change and its regulation is achieved mainly by post-transcriptional control
mechanisms mediated by various nucleus-encoded ribonucleases. More than 180 ribonucleases are annotated in
Arabidopsis, but only 17 are predicted to localize to the chloroplast. Although different ribonucleases act at different
RNA target sites in vivo, most nucleases that attack RNA are thought to lack intrinsic cleavage specificity and show
non-specific activity in vitro. In vivo, specificity is thought to be imposed by auxiliary RNA-binding proteins, including
members of the huge pentatricopeptide repeat family, which protect RNAs from non-specific nucleolytic attack by
masking otherwise vulnerable sites. RNA stability is also influenced by secondary structure, polyadenylation, and
ribosome binding. Ribonucleases may cleave at internal sites (endonucleases) or digest successively from the 5# or
3# end of the polynucleotide chain (exonucleases). In bacteria, RNases act in the maturation of rRNA and tRNA
precursors, as well as in initiating the degradation of mRNAs and small non-coding RNAs. Many ribonucleases in the
chloroplasts of higher plants possess homologies to their bacterial counterparts, but their precise functions have
rarely been described. However, many ribonucleases present in the chloroplast process polycistronic rRNAs, tRNAs,
and mRNAs. The resulting production of monocistronic, translationally competent mRNAs may represent an
adaptation to the eukaryotic cellular environment. This review provides a basic overview of the current knowledge of
RNases in plastids and highlights gaps to stimulate future studies.
Key words: Chloroplast RNA metabolism, ribonucleases, rRNA processing.
Introduction
Chloroplast gene expression is regulated on transcriptional,
post-transcriptional, and translational levels. The predominant post-transcriptional regulation is exerted both at genespecific and global levels (Monde et al., 2000; Cho et al.,
2009). Chloroplasts have retained some part of the eubacterial RNA degradation system but experienced important
evolutionary changes. Unlike in bacteria, nearly if not all
polycistronic transcripts are processed by endo- and exonucleases (Tables 1 and 2), splicing activities, and editing
events in the chloroplast (Bollenbach et al., 2007; Barkan,
2011). Additionally, transcript half-lives differ dramatically,
from an average of 3–8 minutes in bacteria to several hours
in chloroplasts (Klaff and Gruissem, 1991; Bernstein et al.,
2002). Not only the abundance of mRNAs but also the
availability of other RNA species, like non-coding RNAs,
rRNAs, and tRNAs, is an important parameter in determining translation rates and, therefore, the amounts of
proteins produced. Most RNAs undergo an extensive
maturation process in order to become functional. Processing
and degradation of plastid RNA is mediated by ribonucleases. This offers a means of rapidly adjusting RNA
abundance in response to changing environmental conditions, determining the half-life of individual RNAs and
serving as a tool for quality control. In the annotation of the
Arabidopsis genome deposited in TAIR (the Arabidopsis
information resource, www.arabidopsis.org), a total of 182
gene products are assigned as proteins exhibiting ribonucleolytic activity or containing motifs characteristic for RNases,
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1664 | Stoppel and Meurer
Table 1. Endonucleases in eubacteria and in chloroplasts of Arabidopsis
PPR, pentatricopeptide repeat.
Eubacteria
Function
Architecture
Arabidopsis
chloroplasts
Function
Architecture
CSP41b
(Synechocystis)
?
Rossmann fold
CSP41a
(At3g63140),
CSP41b
(At1g09340)
Rossmann fold
RNase E/G
RNA decay initiation;
processing of RNA
precursors
RNE
(At2g04270)
RNase III
Specific for dsRNA;
Processing and decay
of mRNA and rRNA
Specific for RNA in
RNA/DNA hybrids
Endonuclease and
5#/3# exonuclease
5# Maturation of
tRNA precursors
N-terminal catalytic site
with RNase H and S1 domain,
DNase I-like active site;
C-terminal degradosome
scaffold assembly
RNase III, ds RNAbinding domain
RNA decay? Processing
of 23S precursors?
Enhancement of
plastid-encoded
polymerase?
Endonuclease
?
?
?
Rossmann-like fold;
RNase H domain
Metallo-b-lactamase fold
At1g24090
Up-regulated in
PNPase mutants
Essential for plant
viability
Essential for plant
viability, processes
tRNAs in vitro
Essential for plant
viability, processes
tRNAs in vitro
rps7-ndhB RNA
processing
RNase H domain
RNase H
RNase J1/J2
(B. subtilis)
RNase P
RNase Z
3# Maturation of
tRNA precursors
Ribozyme consisting of
catalytic RNA and
protein co-factor
Metallo-b-lactamase
fold
–
–
–
RNJ (At5g63420)
PRORP1 (At2g32230)
CRR2 (At3G46790)
TRZ2 (At2g04530)
Large and unique N-terminal
extension, addition to S1
domain, no homologies to
degradosome scaffold site.
Metallo-b-lactamase
fold
6 PPR motifs; single
protein without RNA
Metallo-b-lactamase-like
10 PPR motifs
Table 2. Exonucleases in eubacteria and in chloroplasts of Arabidopsis
Eubacteria
Function
Architecture
Arabidopsis
chloroplasts
Function
Architecture
PNPase
3#/5# degradation of
RNA
PNP
(At3g03710)
RNase J
(B. subtilis)
RNase R
5#/3# degradation of
RNA and endonuclease
3#/5# degradation of
rRNA and tRNA
2 RNase PH, 2 RNAbinding (KH and S1);
homotrimer
Metallo-b-lactamase
fold
RNase II/R, S1-like
Polyadenylation and
deadenylation; 3#/5#
degradation of RNA
Essential for plant
viability
Targeted to C and M; 3#/5#
degradation of rRNA
2 RNase PH, S1 RNAbinding;
homomultimer
Metallo-b-lactamase
fold
RNase II/R
RNJ
(At5g63420)
RNR1 (At5g02250)
compared to >20 different RNase genes in Escherichia coli
(Arraiano et al., 2010). Out of these 182 gene products, 17
were predicted to be chloroplast localized, as revealed by
using a combination of five different prediction programs.
This variety of gene products, partially exhibiting sequence
similarities of variable significance to their bacterial ancestors,
makes the search for key players in RNA metabolism very
challenging. Assuming that, as in other systems, plant
ribonucleases have relatively low intrinsic sequence specificity,
there are two possible ways of determining target sequences:
either the enzymes may show a preference for substrates with
certain structural or chemical attributes or their activity may
be constrained by pentatricopeptide repeat proteins (PPRs) or
other sequence-specific factors (Pfalz et al., 2009; Stoppel
et al., 2011). The most prominent and best-analysed chloroplast-localized ribonucleases together with up-to-date research results regarding their roles in the control of transcript
maturation and decay will be presented and discussed here.
Endonucleases
Endonucleases (Tables 1 and 3) have various functions in
RNA metabolism. These include maturation of rRNA and
tRNA precursors, processing of polycistronic mRNAs into
monocistronic transcripts, and initiation of RNA decay by
cleavage at exposed sites where no ribosomes or protective
proteins are present. In plant chloroplasts, the main enzyme
responsible for the initial cleavage in the course of RNA
RNases in chloroplasts | 1665
Table 3. Other ribonucleases with predicted chloroplast localization in Arabidopsis
1 ChloroP; 2 SLP; 3 Predotar; 4 TargetP; 5 Wolf Psort; C chloroplast; M mitochondrion.
Function
Locus
1
2
3
4
5
Name
Remarks
RNase H
RNase H
RNase III
C
C
C
C
C
C
M
C
C
C
C
C
C
C
C
–
–
–
RNase III
At1g24090
At5g61090
At1g55140/
At3g13740
At3g20420
C
C
–
C
C
RTL2
Endonuclease
At3g20390
C
C
C
C
C
L-PSP
RNase PH
At4g27490
–
C
C
–
C
–
Exonuclease
Exonuclease
At3g12410
At3g15140
–
C
C
C
C
C
–
C
C
C
–
–
C-terminal archaeal-like RNase HI domain
RNase HII domain
duplicated genes, 70% sequence identity;
Ribonuclease III C terminal domain (RIBOc)
RNase III multi-domain, double-strand RNA
binding motif (DSRM); proposed localization to
nucleus/cytoplasm (Comella et al., 2008)
homologue of rat liver perchloric acid-soluble
protein (L-PSP); chorismate mutase-like
similar to exosome complex poly(A)-dependent
exonuclease RRP41 (Chekanova et al., 2000)
DEDDy 3# 5# exonuclease
DEDDh 3# 5# exonuclease
degradation has yet to been identified. Ribonucleases E and
J and the Rossman fold proteins CSP41a and CSP41b are
among the candidates that have been proposed. However,
since all of the ribonuclease mutants analysed to date fail to
accumulate RNA in significantly greater amounts, none of
them can be unequivocally regarded as rate-limiting for
RNA decay. Processing at specific target sites might also be
mediated by enzymes like RNases E and J (see below). In
contrast to bacteria, in which the synthesis of most proteins
is programmed by polycistronic mRNAs, in chloroplasts,
multi-gene transcripts are often processed in order to
produce translational competent mRNAs.
plastid-encoded polymerase, which would stimulate both
transcription and translation, and a role in stabilizing
intermediates in ribosome assembly have also been discussed
(Bollenbach et al., 2009). Recently, it was proposed that, in
contrast to the endonucleolytic function in vitro, Arabidopsis
CSP41 complexes stabilize non-translated target mRNAs
and precursor rRNAs during the night in vivo, indicating
that defects of csp41 mutants in translation and transcription
are rather secondary effects caused by decreased transcript
stability (Qi et al., 2011).
CSP41a and CSP41b
RNase E and its truncated form, RNase G are well-studied
enzymes in prokaryotes, where RNase E in particular plays
a major role in post-transcriptional regulation of gene
expression by initiating RNA degradation and by mediating
the processing and maturation of rRNAs and tRNAs. In
E. coli, RNase E forms part of a multi-protein complex
called the degradosome, which harbours RNase E, PNPase,
Rhl B, and enolase as major components (Carpousis, 2007).
Bacterial RNase E itself is a large multi-domain protein
with an N-terminal catalytic domain, while its C-terminal
region is responsible for RNA binding and the protein–
protein interactions (degradosome scaffold) that guide
RNase E to its targets. Genes for RNase E homologues
can be found in all higher plant genomes but they differ
from their bacterial ancestors in retaining only the N-terminal
catalytic domain (Schein et al., 2008).
The Arabidopsis nuclear genome encodes an RNase E
homologue (At2g04270) that has been characterized in
several recent studies that support its endonucleolytic
function and localization to the chloroplast stroma (Mudd
et al., 2008; Schein et al., 2008). Despite the partial loss of
endonucleolytic activity on certain RNAs in rne mutants,
there is no significant change in steady-state levels of
most RNAs, and mutant plants are still able to grow
photoautotrophically although with severely reduced
growth rates (Walter et al., 2010). Processing of chloroplast
transcripts still occurs in the mutants, but some unprocessed
The homologous proteins CSP41a and CSP41b are unique
to photosynthetic organisms, although Synechocystis and
Nostoc each contain only one homologue, CSP41b (Schuster
and Stern, 2009). Their Rossman fold domains show
similarity to bacterial epimerase/dehydratase enzymes (Baker
et al., 1998). The function of CSP41a as a non-specific,
RNA-binding endoribonuclease, and the fact that CSP41a
mutants in tobacco show a decrease in turnover of certain
RNA transcripts, both led to the suggestion that CSP41a
might be the enzyme that initiates RNA degradation
(Bollenbach et al., 2003). In spinach, it has been proposed
that CSP41a displays a preference for binding the 3# stemloop structures that are produced during transcript processing, which would make it a candidate for degrading mature
transcripts (Yang et al., 1996). Mutants for Arabidopsis
CSP41b were reported to specifically accumulate precursors
of 23S RNA and the enzyme was therefore proposed to
mediate the final step in the maturation of 23S RNA (Beligni
and Mayfield, 2008). Both forms of Arabidopsis CSP41 have
been reported to interact with each other and to be associated with ribosomes (Bollenbach et al., 2009). However, copurification with ribosomes is often adventitious, especially
when the protein in question is highly abundant in chloroplasts, as is the case for CSP41. A possible function of
Arabidopsis CSP41 in enhancing rates of transcription by the
RNase E/G
1666 | Stoppel and Meurer
high-molecular-weight transcripts accumulate. It was proposed that the mutant phenotype results mainly from
defective processing of transcripts coding for ribosomal
proteins (Walter et al., 2010).
RNase G is a shorter form of RNase E found in E. coli
and some other bacteria. In E. coli, it shows 35% sequence
identity to RNase E, but lacks the C-terminal degradosome
scaffold domain. In contrast to RNase E, RNase G is not
essential for E. coli or Synechocystis (Cohen and McDowall,
1997; Rott et al., 2003). Arabidopsis RNase E shares 37%
sequence identity with E. coli RNase E and 34% with E. coli
RNase G, and, like the latter, it lacks the degradosome
assembly domain. It is therefore not clear whether the
plastid RNE originated from an ancestral gene for E or G.
RNase III
Bacterial RNase III (encoded by the rnc gene) is one of the
many ribonucleases that participates in the maturation of
pre-rRNAs by specifically cleaving at double-stranded
structures, thereby releasing fragments that are subsequently processed further to generate mature 16S, 23S, and
5S rRNAs (Robertson et al., 1968; Gegenheimer and
Apirion, 1975). While RNase E is considered to be the ratelimiting ribonuclease in initiating RNA decay in bacteria,
RNase III shows quite similar features in modulating the
stability of several mRNA species (Babitzke et al., 1993).
Members of the RNase III family are widespread in both
prokaryotic and eukaryotic organisms, exhibiting common
structural and functional features (Lamontagne et al.,
2001). Four classes of RNase III proteins can be distinguished based on their domain organization, containing
different numbers and dispositions of RNase III, doublestranded RNA-binding, Drosha, Dicer, and PAZ (Piwi/
Argonaute/Zwille) domains (Arraiano et al., 2010). The
Arabidopsis genome encodes several proteins with similarities to RNase III, which can be divided into the Dicer-Like
(DCL) and RNase-Three-Like (RTL) families. However,
none of these proteins is known to be localized in the
chloroplast. Previously a plant-specific protein containing
two RNase III domains was identified from maize (Watkins
et al., 2007). This protein was named RNC1 and was shown
to be localized in the chloroplast. Despite its homology to
bacterial RNase III, RNC1 (At4g37510) lacks several
amino acid residues crucial for catalysis. Accordingly, it
has no endonucleolytic activity and has been recruited for
splicing of specific chloroplast group II introns (Watkins
et al., 2007). Still, several other as-yet uncharacterized genes
encoding RNase III motifs are found in the Arabidopsis
genome, out of those At1g55140, At3g13740, and
At3g20420 have been discussed as candidates for the RNase
III function in the chloroplast (Watkins et al., 2007;
Sharwood et al., 2011). However, At3g20420 (RTL2),
despite the predicted chloroplast localization by nearly all
used prediction programs (see Table 3), has been proposed
to be localized to the nucleus and cytoplasm, where it is
presumably involved in 3# external transcribed spacer
cleavage of the pre-rRNA (Comella et al., 2008).
RNase H
The hydrolytic endoribonuclease RNase H is an Mg2+dependent enzyme that specifically cleaves the RNA strand
of RNA/DNA hybrids produced during chromosome
replication (Condon and Putzer, 2002). RNase H is present
in two isoforms (HI and HII) in bacteria, and E. coli can
tolerate the loss of either one, but not both (Ohtani et al.,
1999). Analogous proteins named H1 and H2 can also be
found in eukaryotes (Cerritelli and Crouch, 2009). The
Arabidopsis genome encodes three proteins with RNase H
domains, two of the archaeal HI type with predicted
localizations to mitochondria (At5g51080) and chloroplasts
(At1g24090), respectively (TargetP, ChloroP), and one with
weak similarities to the HII-type predicted to be localized to
chloroplasts (AT5G61090). The chloroplast-localized isoform HI has been shown to be highly up-regulated in
PNPase mutants (Marchive et al., 2009) but no detailed
characterization of mutants is yet available.
RNase J
RNase J is not found in E. coli, but it is present in other
bacteria such as Bacillus subtilis, particularly in species that
lack RNase E, which suggests that the two functions
overlap. This assumption is supported by in vitro cleavage
assays which have shown the two enzymes to be functionally homologous and both have been implicated in rRNA
maturation as well as in RNA decay (Even et al., 2005).
Although RNase J can potentially replace RNase E in
Chlamydomonas, other organisms such as the cyanobacterium Synechocystis possess the shorter RNase G in addition
to RNase J (Kaberdin et al., 1998), while Arabidopsis
possesses both RNase E and J (Bollenbach et al., 2007).
Based on the results of several proteomic studies (Zybailov
et al., 2008; Olinares et al., 2010), Arabidopsis RNase J
(At5g63420) seems to be quite abundant. It is targeted to
the chloroplast (Schuster and Stern, 2009) and, in contrast
to RNase E, it is essential for embryonic development
(http://www.seedgenes.org). Analyses of B. subtilis RNase J
have revealed a 5#/3# exonuclease activity in addition to
its endonucleolytic function (Mathy et al., 2007). A 5#/3#
pathway for mRNA degradation that probably involves
RNase J1 exists in the chloroplast of Chlamydomonas
(Salvador et al., 2011). Whether this holds for higher plants,
and if so to what extent RNase E and J cooperate in this
process, still has to be elucidated.
RNase P
The endoribonuclease P is an essential enzyme in bacteria
that processes polycistronic precursor tRNAs with or
without preceding initial cleavage by RNase E to generate
mature tRNA 5# ends (Mohanty and Kushner, 2007).
Bacterial RNase P is a ribozyme complex composed of two
subunits: a catalytic ‘P RNA’ (encoded by rnpB) and
a ‘P protein’ co-factor (encoded by rnpA), whereas in
archaea and eukaryotes the ribozymes consist of multiple
proteins with no similarities to their bacterial counterpart
RNases in chloroplasts | 1667
(De la Cruz and Vioque, 2003). An RNase P activity
detected in spinach chloroplasts showed efficient cleavage
of precursor tRNAs in vitro (Thomas et al., 2000).
However, extensive phylogenetic sequence analyses have
identified genes homologous to the bacterial rnpB only in
the glaucophyte Cyanophora paradoxa, the red alga Porphyra purpurea, and the green alga Nephroselmis olivacea,
but not in land plants (De la Cruz and Vioque, 2003).
Therefore the mechanism responsible for the specificity of
RNase P activity in land plants lacking the catalytic RNA
seems to be distinct from that employed in E. coli and,
indeed, the question has been raised whether or not this
plant enzyme can still be referred to as RNase P (Altman
et al., 2000). The human mitochondrial RNase P represents
a new type of RNase P composed only of three PPRdomain-containing proteins called MRPP 1, 2, and 3
(Holzmann et al., 2008). The search for sequence orthologues in Arabidopsis led to identification of three proteins
termed ‘proteinaceous RNase P’ or PRORP 1–3 (Gobert
et al., 2010). Two of them (PRORP 2 and 3) are localized in
the nucleus while the third (PRORP1) is targeted to both
chloroplasts and mitochondria. PRORP1 (At2g32230) is
essential for plant viability and processes tRNA precursors
in vitro, suggesting that PRORP proteins have taken over
the functions of the ancestral bacterial ribonucleoprotein in
plants (Gobert et al., 2010).
RNase Z
In contrast to RNase P, which mediates 5# maturation of
tRNAs, RNase Z catalyses maturation at the 3# end of
tRNA precursors lacking the CCA motif (Pellegrini et al.,
2003). Hence tRNA precursor cleavage by RNase Z generates substrates for the addition of CCA to the 3# end by
tRNA nucleotidyltransferase (Betat et al., 2010). The CCA
sequence is essential for aminoacylation and interaction
with the ribosome upon translation (Deutscher, 1973).
Precursor transcripts that already contain a chromosomally
encoded CCA are processed by various endo- and exonucleases, as is the case for many if not all tRNA transcripts
in E. coli (Mörl and Marchfelder, 2001). RNase Z family
members are highly conserved in all three domains of life
and are normally represented by one or two proteins.
However, Arabidopsis encodes four RNase Z genes (TRZ
1–4), all of which exhibit tRNA 3# processing activity
in vitro (Canino et al., 2009). Three of them are dispensable
for plant viability and are targeted to the cytoplasm or
mitochondria or to both the nucleus and the mitochondria.
Deletion of the fourth, chloroplast-targeted RNase Z enzyme, termed TRZ2 or AthTrzS2 (tRNase Z short form 2),
results in embryo lethality (Canino et al., 2009), thus
confirming the importance of this protein in tRNA cleavage.
PPR proteins with endonuclease activity
Proteins with PPR motifs were shown to be involved in many
steps of chloroplast gene expression, like RNA stabilization
(Prikryl et al., 2011), processing (Meierhoff et al., 2003), and
editing (Robbins et al., 2009). It was proposed that proteins
of a PPR subclass containing DYW motifs might bear the
catalytic function required for RNA editing (Salone et al.,
2007). However, DYW motifs do not always seem to be
essential for editing but rather exhibit endonucleolytic
activity in vitro (Okuda et al., 2009). A correlation between
the in vitro cleavage activity of the CRR2 DYW motif and
the inability of crr2 mutants to process the inter-genic region
of the dicistronic transcript rps7–ndhB suggested a function
as a sequence-specific endonuclease (Hashimoto et al., 2003;
Okuda et al., 2009). Future studies will clarify if other DYWcontaining PPR proteins involved in inter-cistronic processing have similar functions.
Exonucleases
Transcript termini which often result from cleavage by
endonucleases are consecutively hydrolysed by exonucleases
(Table 2). They are involved in RNA turnover as well as in
trimming of transcript ends. Three types of eubacterial
exonucleases can be distinguished: 5#/3# exonucleases like
RNase J; 3#/5# exonucleases like PNPase, RNase R, and
RNase II; and poly(A)-specific 3#/5# exonucleases. RNA
degradation in eukaryotes is achieved mainly at the level of
decapping and deadenylation. Eukaryotic 5#/3# exonucleases act rather unspecific degrading all 5# unprotected
RNAs. Decay from the RNA 3# end is mainly carried out
by the multi-protein exosome complex (Schmid and Jensen,
2008). In contrast, chloroplast exonucleases mostly resemble
their eubacterial counterparts. After initial cleavage by
endonucleases, RNA fragments in eubacteria are cleaved
by PNPase and RNases II/R into oligomers of 2–5
nucleotides in length. These oligomers are subsequently
degraded into single nucleotides by oligoribonuclease (Yu
and Deutscher, 1995). While RNase II has not been
reported to occur in higher plants, an oligoribonuclease
homologue is encoded in the Arabidopsis nuclear genome
(At2g26970), although its subcellular localization is predicted not to be in the chloroplast. Other well-studied
bacterial exonucleases, such as RNases BN, D, and T, have
also not been reported in plants, though several genes with
predicted exonucleolytic function are encoded in the Arabidopsis genome (Table 3). Their products await biochemical
characterization.
PNPase
The widely conserved polynucleotide phosphorylase
(PNPase) is an exoribonuclease mediating global RNA
decay. As part of the degradosome, E. coli PNPases mostly
act as trimers attached to RNase E (Carpousis, 2007).
Under normal conditions, E. coli PNPase is not essential for
viability and can be replaced by RNase II or RNase R
(Awano et al., 2008). In E. coli, PNPase processively
catalyses 3#/5# degradation of single-stranded RNA from
newly exposed 3#-termini usually generated by RNase E
(Cohen and McDowall, 1997). Its activity is inhibited by the
formation of stable stem-loops. Polyadenylation of the
1668 | Stoppel and Meurer
transcript 3# end can help overcome this problem, thus
speeding up RNA degradation (Blum et al., 1999).
Chloroplast PNPase (At3g03710) is composed of two
exoribonucleolytic RNase PH core domains, which are involved in RNA degradation and polymerization, together
with an RNA-binding S1 domain that has a high affinity for
poly(A) tails (Yehudai-Resheff et al., 2003). The RNase PH
core domains have been shown to affect the catalysis of
mRNA and rRNA 3#-end maturation as well as to participate
in RNA degradation through exonucleolytic digestion and
polyadenylation (Germain et al., 2011). Despite its propensity for degrading poly(A) tails (Lisitsky and Schuster,
1999), PNPase can also act as a poly(A) polymerase,
depending on environmental conditions (Yehudai-Resheff
et al., 2001). In contrast to eubacteria, the Arabidopsis
PNPase most probably acts as a dimer of trimers without
any other associated proteins (Baginsky et al., 2001). Plants
lacking chloroplast PNPase can still grow photoautotrophically but accumulate RNAs with 3# extensions due to
incorrect end-trimming (Yehudai-Resheff et al., 2001; Walter
et al., 2002). In addition to its role in RNA metabolism,
a function for PNPase in the response to phosphorus
deprivation has been proposed (Marchive et al., 2009).
RNase J
As discussed above, there is strong evidence for the
existence of 5#/3# exonuclease activities in chloroplasts
(Lezhneva and Meurer, 2004; Barkan, 2011). The protein
responsible for this activity has yet not been identified but is
likely to be RNase J, which, like the B. subtilis RNase J1
and J2 orthologues, is likely to possess not only endonucleolytic but also an exonucleolytic activity (Mathy et al.,
2007). A similar exonucleolytic pathway has been shown to
exist in Chlamydomonas (Salvador et al., 2011).
RNase R
Both RNase R and RNase II are 3#/5# hydrolytic
exoribonucleases of the RNase II family. In contrast to
Chlamydomonas, higher plants do not possess any homologue of RNase II, which is thought to act primarily on
mRNAs in bacteria. Bacterial RNase R operates in RNA
quality control, degrading defective tRNAs and rRNAs in
concert with PNPase (Cheng and Deutscher, 2003). Of the
three nucleus-encoded RNase R genes (RNR1–3) in Arabidopsis, RNR1 (At5g02250) was proposed to be targeted to
both chloroplasts and mitochondria, although this has not
been definitively proven (Kishine et al., 2004; Perrin et al.,
2004; Bollenbach et al., 2005). Homozygous Arabidopsis
rnr1 knockout mutants are seedling-lethal, show processing
abnormalities, and accumulate precursors of all ribosomal
RNAs in chloroplasts when grown on sucrose-supplemented
medium (Bollenbach et al., 2005). However, it is not yet
clear whether the phenotypes associated with the mutation
are due to defective rRNA processing alone, since many
chloroplast mRNAs also accumulate to higher levels in rnr1
mutants than in the wild type (Kishine et al., 2004). Defects
in plant growth and development could be explained by
a decrease in accumulation of mature chloroplast ribosomal
RNAs, resulting in a reduced number of ribosomes and
thus reduced translation rates. Furthermore, RNR1 has a
specific role in regulating 5S rRNA levels insofar as it
degrades the complementary 5S antisense RNA (AS5)
which otherwise would form duplex RNAs protecting the
5S RNA from maturation by single-strand-specific endonucleases like RNase E and J (Sharwood et al., 2011). These
duplex RNAs may, in turn, be degraded by a dsRNAspecific ribonuclease such as RNase III.
Other ribonucleases with putative chloroplast
localization
The above-described ribonucleases reflect the current
knowledge of all described RNA cleaving enzymes in
Arabidopsis chloroplasts. To investigate whether additional
previously uncharacterized ribonucleases are present in the
chloroplast, we performed a database search against endo-,
exo-, and ribonucleases based on the Gene Ontology rules
using the annotation of the Arabidopsis genome deposited
in TAIR. A total of 182 genes predicted to encode proteins
with putative ribonuclease motifs were found. Proteins with
just RNA binding properties were left out and candidate
genes were searched carefully for conserved domains known
to be characteristic for ribonucleases. Using five prediction
programs (ChloroP, www.cbs.
dtu.dk/services/ChloroP; SLP, http://sunflower.kuicr.kyoto-u.
ac.jp/;smatsuda/slplocal; Predotar, http://urgi.versailles.inra.
fr/predotar/; TargetP, www.cbs.dtu.dk/services/TargetP; Wolf
Psort, http://wolfpsort.org/), the subcellular localization of all
182 ribonucleases was estimated. Among the previously
characterized endo- and exonucleases (Tables 1 and 2), nine
additional ribonucleases were identified to be chloroplast
localized using a threshold of ‘3 out of 5’ predictor combinations (Table 3). A similar threshold was shown to be both
sensitive and specific to predict organellar localization, as
reflected by Matthews correlation coefficient (Richly and
Leister, 2004). Among these are six endonucleases, two with
predicted RNase H domains and three with RNase III
domains (Table 3). Additionally an L-PSP endonuclease was
identified, which is a homologue of the rat liver perchloric
acid-soluble protein. This L-PSP endonuclease has been characterized as a unique protein not resembling any characteristics to other ribonucleases and was shown to inhibit protein
synthesis by cleaving mRNA in rats (Morishita et al., 1999).
Furthermore, three as-yet uncharacterized exonucleases likely
to be located in chloroplasts were identified. At4g27490 is an
exonuclease showing high similarity to the cytoplasmic
RRP41 protein in Arabidopsis and comprising two RNase
PH domains. RRP41 was shown to be a poly(A)-dependent
exonuclease of the exosome complex (Chekanova et al.,
2000). Two other exonucleases, At3g12410 and At3g15140,
exhibit similarities to DeDDh and DEDDy 3#/5# exonucleases, respectively (Table 3). The identification of uncharacterized ribonucleases is certainly based only on annotations
and a functionality has not been proven experimentally.
Future research will surely clarify this question.
RNases in chloroplasts | 1669
Ribonucleases and rRNA maturation
Chloroplast ribosomes comprise two subunits of 50S and
30S, which together form the 70S ribosome that decodes
mRNAs and translates them into the appropriate polypeptide chains (Harris et al., 1994). Ribosomal subunits are
composed of more than 50 ribosomal proteins (Yamaguchi
and Subramanian, 2000; Yamaguchi et al., 2000), together
with four ribosomal RNAs that are encoded in one gene
cluster, and have been proposed to play a role in the catalytic
activity of the ribosome (Nissen et al., 2000). In spite of the
vast evolutionary distance, the chloroplast rRNA gene
cluster still resembles that of bacteria in terms of organization of coding sequences and co-transcription of genes
(Strittmatter and Kössel, 1984). The clusters in eubacteria
encode mature rRNAs of 16S, 23S, and 5S, while those of
eukaryotes specify 18S, 5.8S, 28/25S, and 5S rRNAs (Fig. 1).
Eukaryotic 5.8S and 28/25S correspond to the 23S rRNA of
eubacteria (reviewed in Evguenieva-Hackenberg, 2005). In
plastids, homologues of eubacterial 23S rRNA are split into
23S and 4.5S, the latter sharing high homology with the 3#
end of the bacterial 23S. In the plastid genome, 16S and 23S
sequences flank a region encoding tRNAs for isoleucine and
alanine and an additional tRNA for arginine is encoded
downstream of the 5S rRNA (Fig. 1).
Ribosomal RNA abundance and processing is of great
importance for ribosome assembly. Thus bacterial cells
usually have multiple copies of the ribosomal RNA operon,
located in different parts of the genome and arranged in a
similar but not identical manner. Accordingly rRNA operons
in eukaryotic nuclear genomes cluster in large tandem
regions containing up to 400 copies in human and many
thousands in some plant species (Long and Dawid, 1980). By
contrast, there are only two rRNA operons in the chloroplast
genome, which are located in the inverted repeats. Maturation of rRNA precursor transcripts performed by endo- and
exoribonucleases is a prerequisite for accurate chloroplast
ribosome biogenesis. Furthermore, some parts of chloroplast
rRNA processing occur in an assembly-assisted manner.
In E. coli, mutants defective in pre-rRNA processing have
been used to elucidate the process of ribosomal RNA
maturation (Gegenheimer and Apirion, 1981). It was shown
that RNase III, in concert with RNase E and G, is
responsible for the endonucleolytic cleavages, while RNase
T performs the exonucleolytic 3# trimming step (Davies
et al., 2010). In vascular plant chloroplasts, several mutants
with putative defects in rRNA processing and ribosome
assembly have been identified to date (Bellaoui et al. 2003;
Bisanz et al. 2003; Bollenbach et al. 2005; Komatsu et al.,
2010; Nishimura et al., 2010; Lu et al., 2011). However, the
precise mechanism of rRNA maturation in chloroplasts
remains an open question, since a biochemical characterization of mutants is often lacking and secondary effects
cannot always be excluded.
Fig. 1. Maturation of rRNA precursors in eukaryotes, eubacteria, and chloroplasts. In eukaryotes, the rRNA genes are arranged in
clusters that fall into two classes. The 28/25S, 18S, and 5.8S rRNAs are cleaved from an 18S–5.8S–28/25S precursor transcript
transcribed by RNA polymerase I, while the 5S rRNA genes form separate clusters that are transcribed by RNA polymerase III (Haeusler
and Engelke, 2006). The bacterial rrnC operon shown here is one of seven rRNA operons in the E. coli genome. The primary transcript is
first cleaved by RNase III and the resulting intermediates are then trimmed by the RNases E, G, and T (Davies et al., 2010). The RNases
(arrows) responsible for maturation of the primary transcript in chloroplasts are still unknown. It has been suggested that tRNAArg is
cleaved by the RNases P and Z, and 5# maturation of 23S rRNA has been attributed to CSP41a/b. RH39 was proposed to function in
cleavage of the second ‘hidden break’ (triangle). Endonucleolytic cleavage intermediates are subsequently trimmed at their 3# ends by
PNPase and/or RNase R (grey) and possibly also at their 5#-ends by an exonuclease that could be RNase J (black).
1670 | Stoppel and Meurer
In chloroplasts, the 7.4 kb rRNA precursor is thought to
be cleaved by an as-yet unidentified endonuclease, releasing
pre-tRNAs for isoleucine and alanine and pre-rRNAs
for 16S, as well as the dicistronic intermediates 23S–4.5S
and 5S–tRNAArg (Fig. 1). The pre-tRNAs are subsequently
processed at their 5# and 3# ends by RNase P and RNase Z,
respectively (Canino et al., 2009; Gobert et al., 2010). The
processing intermediate 23S–4.5S is first matured at the 23S
5# and 4.5S 3# end, and then endonucleolytically cleaved in
several steps at the 5# end of 4.5S. Inter-cistronic cleavage
requires both assembly of the dicistron into pre-ribosomal
subunits and prior 3#-end maturation of 4.5S rRNA
(Bellaoui et al. 2003). Mutants defective in rRNA 3#
processing or ribosome assembly accumulate this 23S–4.5S
processing intermediate (Bellaoui et al. 2003; Bisanz et al.
2003; Bollenbach et al. 2005). The subsequent 3#-end
trimming of 23S rRNA is performed by PNPase and
RNR1 (Yehudai-Resheff et al., 2001; Bollenbach et al.
2005). The mature 23S rRNA is then cleaved internally at
so-called ‘hidden breaks’, as revealed by electrophoresis on
agarose gels under denaturing conditions (Leaver, 1973).
Although a DEAD-box helicase with a proposed function
in formation of one of the hidden breaks was recently
identified (Nishimura et al., 2010), the need for such
additional modifications of rRNAs in the large ribosomal
subunit is not understood.
In contrast to bacteria, precursors of chloroplast 16S
RNA are not processed close to their mature termini. Thus
they possess long 3# tails requiring 3#/5# exonucleolytic
processing which is performed, as in the case of 23S, by the
RNase R homologue RNR1 and/or PNPase, as reported
previously (Yehudai-Resheff et al., 2001; Bollenbach et al.
2005). The 5S RNA is co-transcribed with the downstream
tRNAArg in Brassica and Arabidopsis (Leal-Klevezas, 2000;
Sharwood et al., 2011) followed by endonucleolytic cleavage
and exonucleolytic 3#-end trimming.
Regulation of site specificity and transcript abundance
Various mechanisms can determine the stabilities of chloroplast mRNAs, including protection of RNA termini by
proteins or RNA secondary structures. Since untranslated
regions are not protected by ribosomes, they are typical sites
of endonucleolytic cleavage by RNase E/J or CSP41, making
accessibility to such sequences a key determinant of mRNA
stability. Recently a PPR protein, PPR10, was reported to
serve as a barrier to 5#- and 3#-end exonucleolytic RNA
decay, providing an alternative to stabilizing RNA stem-loop
structures (Pfalz et al., 2009). A similar observation has been
made regarding the protein PrfB3, which originated from
a ribosomal release factor. PrfB3 stabilizes the 3# ends of
petB transcripts, most likely by protecting them from
exonucleolytic digestion (Stoppel et al., 2011). Light- and
stress-dependent control of PrfB3 levels likewise allows the
plant to adjust cytochrome b6 levels, which determine overall
rates of photosynthesis (Stoppel et al., 2011). Thus binding of
protective proteins at both the 5# and 3# ends can define
chloroplast RNA transcript termini and abundance without
the need for sequence specificity of ribonucleases. For
example, endonucleases like CSP41 and RNase E were shown
to cleave RNA without any specificity in vitro (Yang et al.,
1996; Schein et al., 2008). But while RNases E and J are
postulated to cleave rather non-specifically in vivo as well,
giving exonucleases access to internal RNA regions, CSP41a
displays a preference for RNAs containing hairpin structures,
making it a candidate for determining transcript half-life
in the chloroplast (Bollenbach et al., 2003). Among the
exonucleases, PNPase preferentially degrades polyadenylated
sequences (Lisitsky and Schuster, 1999), while the activity of
RNR1 could, like that of the E. coli RNase II (Coburn and
Mackie, 1996), be modulated by RNA secondary structures
in vivo. An interaction with other proteins like helicases is
also conceivable, as it would facilitate the cleavage of highly
structured substrates, as is the case for the E. coli degradosome, in which PNPase is associated with the RNA helicase
RhlB. Last but not least, translational events have been
shown to influence RNA stability (Meurer et al., 2002).
Conclusions
Ribonucleases have long been considered as purely degradative enzymes, cleaving without any sequence specificity.
But nowadays a greater awareness of the significance of
both endo- and exoribonucleases is emerging, in particular
because it has become apparent that many RNases can
display specificity for sequences or structures, allowing them
to regulate transcript abundance according to the needs of
the cell. It is becoming obvious that RNases play a central
role in RNA metabolism, including RNA decay, maturation of RNA precursors, and end-trimming of certain
RNAs. However, a truly regulatory role for RNases has yet
to be convincingly shown. As discussed here, RNases
contribute to the implementation of molecular processes in
response to environmental signals, emphasizing the importance of RNA metabolism in adjusting chloroplast functions. A single cell or organelle can contain various RNases
with sometimes overlapping functions and specificities, and
high-molecular-weight complexes function together with
stabilizing elements such as PPRs in order to control overall
RNA accumulation. Increasing knowledge of the functional
features of ribonucleases inside the chloroplast will become
more and more important, since organelles are advancing as
objectives for plant transformation.
Acknowledgements
The authors wish to thank the Deutsche Forschungsgemeinschaft for support (grant number SFB TR1).
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