Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
The mRNA Closed-loop Model: The Function of PABP
and PABP-interacting Proteins in mRNA Translation
A. KAHVEJIAN, G. ROY,
AND
N. SONENBERG
Department of Biochemistry and McGill Cancer Center, McGill University, Montréal, Québec, Canada
Translational control is an important means by which
cells govern gene expression. It provides a rapid response
to growth and proliferation stimuli and plays a role in
controlling feedback mechanisms in the cell. In systems
with little or no transcriptional control (e.g., reticulocytes
and oocytes), translation is the predominant mode of regulation of gene expression (for review, see Mathews et al.
2000). Initiation, the rate-limiting step of translation, is
often the target of translational control. All nuclear-transcribed eukaryotic mRNAs possess a 5´ cap (m7GpppN,
where m is a methyl group and N is any nucleotide), and
most possess a 3´ poly(A) tail. The focus of this paper is
the role of the poly(A) tail and the poly(A)-binding protein (PABP) in translation initiation. Such an analysis requires a brief introduction of translation initiation and its
control.
Initiation of translation consists of recruitment of the
ribosome to mRNA, negotiation of the 5´-untranslated region (5´UTR), and recognition of an initiation codon (for
review, see Hershey and Merrick 2000). Eukaryotic
translation initiation factors (eIFs) mediate the ribosome
recruitment process. The eIF4 family of initiation factors
is responsible for the recruitment of the small 40S ribosomal subunit to the 5´ cap structure. The cap is recognized by the eIF4F complex, which consists of eIF4E,
eIF4A, and eIF4G. eIF4E binds directly to the mRNA 5´
cap structure, eIF4A is an RNA helicase, and eIF4G is a
modular scaffolding protein that binds eIF4E, eIF4A,
eIF3, PABP, and Mnk, a Ser/Thr kinase that phosphorylates eIF4E (for review, see Gingras et al. 1999; Pyronnet
et al. 1999). eIF3 also interacts with the 40S ribosomal
subunit, thus serving as a link between the mRNA/eIF4F
complex and the ribosome. eIF4A, in conjunction with
eIF4B and eIF4H, is thought to unwind the mRNA 5´ secondary structure to facilitate 40S ribosome binding (for
review, see Hershey and Merrick 2000). eIF4A may also
be involved in the progression of the 40S ribosomal subunit along the mRNA 5´UTR. Upon recognition of an initiation codon, most of the initiation factors are released,
followed by the joining of the large 60S ribosomal subunit (for review, see Hershey and Merrick 2000). Ribosomes can also be recruited to the mRNA via an alternate
pathway through an internal ribosome entry site (IRES).
A number of viral RNAs and cellular mRNAs contain
IRESs, in their 5´UTR, which bypass the requirement for
a cap structure for translation initiation. Certain IRESs
interact with translation factors (e.g., the encephalomyocarditis virus IRES; Lomakin et al. 2000), whereas others directly interact with the 40S ribosomal subunit (e.g.,
the hepatitis C virus IRES; Kieft et al. 2001).
There are numerous examples of translational control
which are exerted at the ribosome recruitment step, both
general (applying to large numbers of mRNAs) and
mRNA-specific. Notably, cap-dependent translation is
modulated by the eIF4E binding proteins (4E-BPs). These
small repressor molecules interact with eIF4E to compete
with eIF4G binding, and thus inhibit eIF4F complex formation and initiation of translation (for review, see Raught
et al. 2000). This inhibition is reversible, as the affinity of
4E-BPs for eIF4E is modulated by 4E-BP phosphorylation. Phosphorylation is regulated by many types of extracellular stimuli, including growth factors, hormones, and
mitogens, all of which activate the PI3-kinase signaling
pathway (for review, see Gingras et al. 2001). This mechanism highlights the importance of the link between extracellular stimuli and translational control.
Regulation of translation may also be mediated via the
mRNA 3´UTR. For instance, short-lived mRNAs, which
code for proto-oncogene products, cytokines, and early
response gene products, contain AU-rich elements (ARE)
in their 3´UTR which, under most circumstances, confer
mRNA instability (Chen and Shyu 1994; Stoecklin et al.
1994; Xu et al. 1997). However, for some mRNAs the
ARE affects translation. For example, the mRNA encoding tumor necrosis factor-α (TNF-α) contains an ARE
that represses translation (Gueydan et al. 1999; Piecyk et
al. 2000). TNF-α translation is stimulated in macrophages in response to lipopolysaccharides (LPS), by a
mechanism that involves the removal of a repressor protein from the ARE (Piecyk et al. 2000). A large number
of mRNAs contain 3´UTR elements that regulate translation during early development; the majority of these studies have been conducted in Drosophila (for review, see
Wickens et al. 2000). The formation of the caudal protein
gradient in the Drosophila embryo is dependent on a bicoid-response element (BRE) in the 3´UTR of the caudal
mRNA. The bicoid protein interacts with the BRE and
suppresses caudal mRNA translation (for review, see
Wickens et al. 2000). Another example is the 3´UTR of
15-lipoxygenase (LOX), which contains a differentiation
control element (DICE). When bound by heterogeneous
nuclear ribonucleoproteins K and E1 (hnRNP-K and
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXVI. © 2001 Cold Spring Harbor Laboratory Press 0-87969-619-2/01.
293
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
294
KAHVEJIAN, ROY, AND SONENBERG
hnRNP-E1), this element represses translation (Ostareck
et al. 1997). Recent studies have demonstrated that the
DICE inhibits 60S subunit joining, rather than 40S recruitment (Ostareck et al. 2001). This mechanism is unexpected, as regulation of translation initiation usually
occurs at the 40S ribosome recruitment step.
How mRNA 3´UTR elements may regulate translation
is not well understood, but the recent findings demonstrating the circularization of eukaryotic mRNAs (see below) provide a possible framework to explain their mechanism of action. A key player in this mechanism is the
poly(A) tail, which is bound by PABP. Here, we describe
the mechanism by which PABP stimulates translation,
and the various factors involved in this process, including
two proteins recently identified in our laboratory.
THE POLY(A) TAIL AND TRANSLATION
The function of the poly(A) tail has been an active area
of research for three decades (Kates 1971; Adesnik et al.
1972). Early in vitro experiments suggested a role for the
poly(A) tail in translation initiation. The poly(A) tail was
demonstrated to confer a translational advantage to the
mRNA in reticulocyte lysate, and addition of poly(A)
RNA inhibited the translation of poly(A)+ mRNA (Doel
and Carey 1976; Jacobson and Favreau 1983; Grossi de Sa
et al. 1988). More detailed experiments comparing the
translatability, degradation, polysome profile, and assembly into messenger ribonucleoprotein complexes
(mRNPs) of synthetic poly(A)+ versus poly(A)– mRNA
were carried out in a reticulocyte lysate translation system
by Munroe and Jacobson (1990b). These studies demonstrated a two- to threefold translational stimulation conferred by the poly(A) tail, which was not attributable to its
mRNA-stabilizing effect (Munroe and Jacobson 1990b).
Furthermore, addition of exogenous poly(A) to these extracts inhibited the translation of poly(A)+ mRNA, while
stimulating the translation of poly(A)– mRNA. Another
finding consistent with the poly(A) tail´s importance in
translation is the positive correlation between the
polyadenylation state of an mRNA and its translational activation during development. In many systems (e.g.,
Xenopus, Drosophila, mouse), the translation of maternal
mRNAs in the oocyte coincides with their polyadenylation state (for review, see Wickens et al. 2000).
PABP
PABP was discovered in 1973 as the major protein associated with the poly(A) tail of eukaryotic mRNAs (Blobel 1973). Numerous studies in yeast have implicated
PABP in mediating the effects of the poly(A) tail on
translation initiation. Depletion of PABP in yeast by promoter inactivation or use of a temperature-sensitive mutation reduced translation initiation and cell growth
(Sachs and Davis 1989), whereas immunodepletion of
PABP from yeast extracts repressed translation of
polyadenylated mRNAs (Tarun and Sachs 1995). PABP
is an essential protein, because in yeast, deletion of the
PAB1 gene is lethal (Sachs et al. 1987).
PABP is a relatively abundant protein; HeLa cells contain approximately six molecules per ribosome (Görlach
et al. 1994). It is a multidomain mRNA-binding protein
containing four phylogenetically conserved RNA recognition motifs (RRMs) (Adam et al. 1986; Sachs et al.
1987). The RRM is the most common RNA-binding domain, and exists in more than 200 putative RNA-binding
proteins (for review, see Nagai 1996; Varani and Nagai
1998; Perez-Canadillas and Varani 2001). X-Ray crystallography studies have demonstrated that the RRM consists of four antiparallel β sheets flanked by two α helices
(Oubridge et al. 1994). Two conserved sequence motifs,
RNP1 and RNP2, are located in the central two β strands
and make contact with the RNA (Nagai 1996; Deo et al.
1999). PABP fragments containing RRMs 1 and 2 bind
poly(A) RNA with an affinity similar to wild-type. RRMs
3 and 4 display a tenfold lower affinity for poly(A) (Burd
et al. 1991; Kuhn and Pieler 1996; Deo et al. 1999). The
proline-rich, carboxy-terminal one-third of the protein
serves as a docking site for a number of proteins, including PABP interacting proteins 1 and 2 (Paip1 and Paip2)
(see below; Deo et al. 2001; Khaleghpour et al. 2001b;
Kozlov et al. 2001).
Different fragments of PABP can individually stimulate translation in Xenopus oocytes, independently of
their poly(A)-binding activity (Gray et al. 2000). A fragment containing RRMs 1 and 2 of PABP, which binds
eIF4G (see below), is more effective than full-length
PABP in stimulating translation (Gray et al. 2000). A
fragment consisting of RRMs 3 and 4 or the carboxyl terminus of PABP can also stimulate translation, albeit to a
lesser degree (Gray et al. 2000). Otero et al. (1999)
demonstrated that exogenous PABP stimulates the translation of capped poly(A)+ and, to a lesser extent, poly(A)–
mRNA in yeast extracts via distinct mechanisms. These
findings suggest that the mechanism by which PABP
stimulates translation is complex and may involve redundant or alternative pathways.
TRANSLATIONAL SYNERGY BETWEEN
THE 5´ CAP AND THE POLY(A) TAIL
Suggestive evidence for the circularization of mRNA
existed long before the discovery of the underlying protein interactions responsible for bridging the 5´ and 3´ termini (see below). The closed-loop model for mRNA circularization was proposed almost two decades ago
(Jacobson and Favreau 1983; Palatnik et al. 1984) and reiterated in the following years (Sachs and Davis 1989;
Munroe and Jacobson 1990a,b; Jacobson 1996). Furthermore, electron micrographs of rat pituitary cell cross-sections revealed a predominance of circular polysomes on
the surface of the rough endoplasmic reticulum (Christensen et al. 1987). The synergistic enhancement of translation observed in mRNAs containing both a 5´ cap and
3´ poly(A) tail also suggested a physical interaction between the mRNA extremities (Gallie 1991). Electroporation of mRNAs into cells demonstrated that those possessing both a cap and a poly(A) tail were more
efficiently translated than mRNAs possessing only one of
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
CLOSED-LOOP MODEL OF TRANSLATION
the structures, and that this enhancement was synergistic
(Gallie 1991). Capped and polyadenylated mRNAs display similar synergistic properties in yeast extracts
(Iizuka et al. 1994), indicating that the poly(A) tail plays
an important role in stimulating cap-dependent translation initiation.
THE eIF4G/PABP INTERACTION
The synergism between the mRNA 5´ and 3´ ends
could be readily explained if they were to physically interact. Proof of a direct interaction between the 5´ and 3´
ends of mRNA proved elusive until the discovery of the
physical association between eIF4G and PABP in yeast
(Tarun and Sachs 1996) and plant systems (Le et al.
1997). However, at the same time, an interaction between
mammalian PABP and eIF4G could not be demonstrated
(see e.g., Craig et al. 1998). The subsequent discovery of
an extended amino terminus of human eIF4G explains
this difficulty, as it revealed the binding site for PABP
(Imataka et al. 1998; Piron et al. 1998). A stretch of 29
amino acids in the amino terminus of human eIF4G interacts with RRMs 1 and 2 of human PABP (Imataka et
al. 1998), as in yeast PABP. However, despite its high homology with yeast PABP, human PABP does not interact
with yeast eIF4G (Otero et al. 1999), likely because the
PABP-binding sites in the human and yeast eIF4G proteins are quite divergent. The fact that this interaction has
been conserved through evolution (or arose twice), despite the divergence of the protein sequences, underscores its functional significance.
The interaction between PABP and eIF4G results in the
circularization of the mRNA. Indeed, atomic force microscopy experiments demonstrated the formation of
RNA circles when recombinant yeast eIF4G, eIF4E, and
PABP were mixed with an mRNA possessing a 5´ cap
and a 3´ poly(A) tail (Wells et al. 1998). The absence of
any of the proteins prevented circle formation, as did the
use of eIF4G mutants defective in eIF4E or PABP binding (Wells et al. 1998).
mRNA CIRCULARIZATION:
BIOLOGICAL SIGNIFICANCE
What is the purpose of mRNA circularization? An interesting idea is that circularization permits efficient
translation of intact mRNAs only, thus diminishing the
possibility of generating potentially dominant negative
forms of proteins from nicked mRNAs. The biological
significance of circularization has been studied in several
systems. Surprisingly, yeast strains containing an eIF4G
mutant defective in PABP binding are viable, unless
combined with a mutation in eIF4E (Tarun et al. 1997).
This synthetic lethality suggests a redundancy in PABP
and eIF4E function (Tarun et al. 1997). In contrast, the
eIF4G–PABP interaction appears to play a critical role in
Xenopus oocytes, because expression of an eIF4G mutant
that does not bind PABP repressed translation of
polyadenylated mRNAs and inhibited progesterone-
295
induced oocyte maturation (Wakiyama et al. 2000). Recently, the interaction between eIF4G and PABP was
shown to be required for translational control of ceruloplasmin (Cp) (Mazumder et al. 2001). Binding of cytosolic factors to the 3´UTR of Cp mRNA represses translation by a mechanism dependent on circularization;
immunodepeletion of PABP or eIF4G, or the absence of
a poly(A) tail, abrogated the translational silencing of Cp
mRNA (Mazumder et al. 2001).
5´–3´ interactions also occur in viral mRNAs lacking a
cap structure or a poly(A) tail. Picornaviral RNAs do not
possess a 5´ cap structure, and they initiate translation via
ribosome binding to an IRES (Belsham and Jackson
2000). In this case, the poly(A) tail and the IRES synergistically promote translation, which may be explained
by the direct binding of eIF4G to both the IRES and
PABP (Bergamini et al. 2000; Lomakin et al. 2000). Hepatitis C virus (HCV) RNA also contains an IRES, but no
poly(A) tail, and does not require eIF4G for translation
(Kieft et al. 2001). In a model proposed by Ito and Lai, the
polypyrimidine-tract-binding protein (PTB) serves to circularize HCV RNA and thus enhance its translation by interacting with an element in the 3´UTR and to a protein
that interacts with the HCV IRES (Ito and Lai 1999).
Strong evidence for the functional requirement of circularization exists for rotavirus mRNAs, which are capped
but not polyadenylated. These mRNAs contain a conserved sequence (UGACC) in their 3´UTR that is recognized by the viral NSP3 protein (Poncet et al. 1993).
NSP3 interacts with the amino terminus of eIF4G to stimulate viral mRNA translation (Poncet et al. 1993, 1996;
Piron et al. 1998; Vende et al. 2000). In addition, NSP3
displaces PABP from eIF4G and thus inhibits host protein synthesis (Piron et al. 1998). These findings ascribe
a key role for PABP as a translation factor and stress the
importance of circularization for translational activation.
MECHANISM OF PABP STIMULATION OF
TRANSLATION INITIATION
Several models have been proposed to explain the
mechanism by which circularization promotes translation. First, circularization could promote the recycling of
terminating ribosomes on the same mRNA by bringing
the two ends of the mRNA together. Another model suggests that PABP increases 60S ribosomal subunit joining.
A third model posits that PABP stabilizes the eIF4F–cap
interaction and thus stimulates 40S ribosomal subunit
binding.
In early experiments, Sachs and Davis observed that
mutations in a 60S ribosomal protein or in a helicase required for 60S ribosomal subunit biosynthesis were capable of rescuing the phenotype of the PABP deletion in
yeast (Sachs and Davis 1989,1990). These genetic data
are supported by biochemical experiments, as Munroe
and Jacobson (1990b) reported that the absence of the
poly(A) tail led to a decrease in 60S ribosomal subunit
joining. Subsequent genetic studies in yeast are also consistent with such a model: Wickner´s group showed that
the poly(A) tail derepresses translation (Widner and
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
296
KAHVEJIAN, ROY, AND SONENBERG
Wickner 1993). Poly(A)+ mRNA lost its translational advantage over poly(A)– mRNA in yeast mutants lacking
two Ski RNA helicase proteins, Ski2p and Slh1p. This is
due to a dramatic increase in the translation of poly(A)deficient mRNA, with no effect on poly(A)+ mRNA
(Widner and Wickner 1993). Deletion of eIF5B or mutation of eIF5, two initiation factors involved in 60S ribosomal subunit joining, drastically reduced translation of
poly(A)+ mRNA, with little effect on poly(A)– mRNA.
Furthermore, the increase in poly(A)– mRNA translation
in the Ski2∆ Slh1∆ strain was lost in an eIF5B null background (Searfoss et al. 2001). The Ski proteins were suggested to act by inhibiting eIF5 and eIF5B, thus impeding
60S ribosomal subunit joining. Importantly, this inhibition is repressed by PABP (Searfoss et al. 2001).
Notwithstanding these data, other experiments argue for
a role of PABP in recruiting the 40S ribosomal subunit to
the mRNA. In extracts immunodepleted of PABP,
mRNA translation and 40S ribosomal subunit recruitment were inhibited (Tarun and Sachs 1995). Biochemical data also suggest that the mRNA 5´–3´ interactions
may stabilize the formation of the 5´ initiation complex.
PABP was reported to increase the affinity of eIF4F for
the cap structure (Wei et al. 1998) and to interact with
eIF4B in plant and mammalian systems (Le et al. 1997;
Bi and Goss 2000; Bushell et al. 2001). One possible explanation for the discrepancy between the models is that
when 60S ribosomal subunit joining is inhibited (as in the
eIF5 or eIF5B mutants, or by diminishing 60S biogenesis), 60S ribosome joining becomes rate-limiting for initiation, and thus the difference between poly(A)+ and
poly(A)– mRNAs in 40S recruitment would be difficult to
detect.
PABP INTERACTING PROTEINS 1 AND 2
Interaction screening using the far western technique
with radiolabeled PABP led our laboratory to the cloning
of two PABP-binding partners: PABP interacting proteins 1 and 2 (Paip1 and Paip2) (Craig et al. 1998;
Khaleghpour et al. 2001a). Paip1 is a 54-kD protein that
stimulates translation in vivo. It shares homology with
the central region of eIF4G, which contains an eIF4Abinding site, and it interacts with eIF4A (Craig et al.
1998). Paip1 is also involved in mRNA turnover. It is
found in a protein complex that stabilizes the c-fos protooncogene mRNA by binding to the major protein-codingregion determinant of instability (mCRD) (Grosset et al.
2000). Conversely, Paip2 (MW = 14 kD) preferentially
inhibits the translation of mRNAs possessing a poly(A)
tail (Fig. 1A) (Khaleghpour et al. 2001a). Translation of
an mRNA containing the HCV IRES is not inhibited by
Paip2 (Fig.1B) (Khaleghpour et al. 2001a), most likely
because this IRES translates independently of eIF4G
(Pestova et al. 1998). Paip2 inhibits 80S ribosome complex formation in a ribosome-binding assay (Khaleghpour et al. 2001a), but its effect on 40S ribosome complex
formation has not been examined. How might Paip2 inhibit ribosome complex formation? Paip2 competes with
Paip1 for binding to PABP, and in an RNA-binding as-
Figure 1. Paip2 inhibits translation in vitro. (A) Translation of
capped poly(A)+ or poly(A)– luciferase mRNA in the presence
of Paip2 in a Krebs-2 cell-free translation system. Shown as relative luciferase activity (% of control). (B) Effect of Paip2 on
translation of capped, poly(A)+ bicistronic CAT-HCV IRES-luc
mRNA in a Krebs-2 cell-free translation system. Shown as relative luciferase activity (% of control). (Adapted, with permission, from Khaleghpour et al. 2001a.)
say, strongly interdicts the interaction of PABP with
poly(A), thus disrupting the poly(A)-organizing activity
of PABP (Fig. 2A, lanes 1–4) (Khaleghpour et al. 2001a).
The simplest model to depict Paip2 function is that it inhibits translation initiation by disrupting the circularization of mRNAs (Fig. 3). The binding sites for Paip2 and
eIF4G on PABP overlap (see below), suggesting that they
compete for binding to PABP. A mutant of Paip2, Paip2
(1-42), which does not bind to PABP, and a mutant that
binds only to the carboxyl terminus, but not to the RRM
region of PABP, Paip2 (76-127), do not affect the
PABP/poly(A) interaction (Fig. 2A, lanes 5–8 and 13–16,
and Fig. 2B). Furthermore, these fragments of Paip2 do
not inhibit translation (Fig. 2C) (Khaleghpour et al.
2001b).
Paip1 interacts with RRMs 1 and 2 and the carboxyl
terminus of PABP (G. Roy et al., unpubl.), whereas Paip2
interacts with RRMs 2 and 3, and the carboxyl terminus
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
CLOSED-LOOP MODEL OF TRANSLATION
297
Figure 3. Model for the inhibition of translation by Paip2. Paip2
inhibits PABP binding to the mRNA and may interfere with the
PABP/eIF4G interaction on the mRNA, resulting in the disruption of the circular conformation.
of PABP (Khaleghpour et al. 2001b). Paip2 binds to
PABP with a 2:1 stoichiometry with two independent Kds
of 0.66 and 74 nM (Khaleghpour et al. 2001b). Paip1
binds to PABP with a Kd of 1.9 nM, and the stoichiometry
of this interaction is currently under investigation. Importantly, the interactions of Paip1 and Paip2 with PABP are
mutually exclusive (Khaleghpour et al. 2001a). The binding of both Paip1 and Paip2 to PABP requires RRM2
(Khaleghpour et al. 2001b; G. Roy, unpubl.), and both
proteins contain a conserved 15-amino-acid stretch necessary for binding the carboxyl terminus of PABP (Fig. 4)
(Khaleghpour et al. 2001b).
NEW PABP INTERACTING PARTNERS
Figure 2. Functional dissection of Paip2. (A) Disruption of
poly(A)-organizing activity of PABP by Paip2 fragments. (B)
Inhibition of PABP binding to poly(A) by Paip2 fragments in a
filter binding assay. (C) Effects of Paip2 fragments on capped
poly(A)+ luciferase mRNA translation in a Krebs-2 cell-free
translation system. (Adapted, with permission, from Khaleghpour et al. 2001b.)
The 15-amino-acid stretch, which is shared by Paip1
and Paip2, is also present in a number of other proteins
(Fig. 4), suggesting that the carboxyl terminus of PABP
has other binding partners and perhaps undiscovered
roles unrelated to translation ( Deo et al. 2001; Kozlov et
al. 2001). Paip1 and Paip2 may therefore compete with
the carboxy-terminal binding partners of PABP and modulate other aspects of PABP function. For example,
eRF3, the eukaryotic translation termination factor, also
contains the conserved 15-amino-acid sequence in its
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
298
KAHVEJIAN, ROY, AND SONENBERG
(Yoshida et al. 2001), an RNA-binding protein involved
in mRNA deadenylation (Tucker et al. 2001). The possible interaction of Tob with the carboxyl terminus of
PABP may implicate Tob in mediating signaling to the
translational and deadenylation apparatus. Paip1 and
Paip2 also interact with the carboxyl terminus of the
HYD ubiquitin ligase via the same 15-amino-acid stretch
(Deo et al. 2001), potentially targeting these proteins for
degradation. It would be of interest to determine whether
ubiquitination serves as a regulator of mRNA circularization and translation.
REGULATION OF Paip FUNCTION
Figure 4. PABP interaction sites. (A) Respective PABP-binding
sites in Paip1 and Paip2. (B) Alignment of the PABP carboxyterminal binding site in Paip1 and Paip2 with amino acid sequences in eukaryotic release factor 3 (eRF3), Ataxin-2, and
Tob. The interactions between the first three proteins in this list
and the carboxyl terminus of PABP have been reported.
amino terminus and interacts with the carboxyl terminus
of PABP (Hoshino et al. 1999). The large distance between the translation termination codon and the poly(A)
tail in mRNAs with long 3´UTRs has served as an argument against the poly(A) tail’s role in ribosome recycling. However, the interaction between eRF3 and PABP
may provide a physical link between a translation-terminating ribosome and the 3´ poly(A) tail for recruitment to
the 5´ end of the mRNA. Another surprising and interesting protein that contains the PABP carboxy-terminal
binding motif, Ataxin-2, is implicated in spinocerebellar
ataxia (Lorenzetti et al. 1997) and interacts with another
RRM-containing protein, A2BP1 (Shibata et al. 2000).
The function of Ataxin-2 is unknown, but its possible interaction with PABP may shed new light on spinocerebellar ataxia. The 15-amino-acid motif is also found in
the transducer of ErbB-2 (Tob) (Deo et al. 2001), which
interacts with p185erbB2, a transmembrane glycoprotein
containing an SH2-binding domain that stimulates cell
growth (Matsuda et al. 1996). Tob has antiproliferative
properties and is homologous to two other suppressors of
cell growth, B-cell translocation gene protein 1 (BTG-1)
and the abundant in neuroepithelium area protein (ANA)
(Yoshida et al. 1998). Members of this family of proteins
also interact with the human homolog of yeast Caf1
Paip2 is a phosphoprotein, the phosphorylation state of
which is modulated by serum (A. Kahvejian and B.
Raught, unpubl.). The functional significance, the intracellular signaling pathways involved in this regulation,
and the locations of the phosphorylation sites are currently unknown. Paip1 is also phosphorylated (B. Raught
and A.-C. Gingras, unpubl.). Does phosphorylation of
Paip1 and Paip2 serve as a switch to promote or inhibit
the circularization of mRNA? Elucidation of the pathways responsible for these modifications and of the biochemical consequences that ensue will help to answer this
question. If phosphorylation indeed modulates Paip1 and
Paip2 function at the 3´ end of the mRNA, the mechanism
would resemble the modulation of 4E-BP function by
phosphorylation (Raught et al. 2000).
CONCLUSIONS
Translational control had long been thought to be mediated primarily via the 5´ end of the mRNA. However,
over the last decade, observations of the effects of the
poly(A) tail and PABP on translation initiation have provided renewed importance to the 3´UTR of mRNA. Yet,
the mechanism of poly(A) action is not well understood.
The discovery of the interaction of PABP with both
eIF4G and the Paips may explain the link between the 5´
and 3´ ends of the mRNA and shed new light on the role
of PABP as a translation factor. It is noteworthy that
Paip1 and Paip2 exist only in metazoans. They may thus
function in a higher-order mechanism of translational
control in the metazoa. Further research into the regulation of these proteins and the discovery of new protein–protein or protein–RNA interactions will result in a
more in-depth understanding of the importance of mRNA
circularization for translation and translational control.
ACKNOWLEDGMENTS
We thank Brian Raught, Francis Poulin, Mathieu
Miron, and Rahul C. Deo for critical reading of this
manuscript. This work was supported by a grant from the
Canadian Institute of Health Research to N.S. N.S. is a
distinguished scientist of the Canadian Institute of Health
Research and a Howard Hughes International Scholar.
A.K. and G.R. were recipients of doctoral studentships
from the Canadian Institute of Health Research.
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
CLOSED-LOOP MODEL OF TRANSLATION
REFERENCES
Adam S.A., Nakagawa T., Swanson M.S., Woodruff T.K., and
Dreyfuss G. 1986. mRNA polyadenylate-binding protein:
Gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 6: 2932.
Adesnik M., Salditt M., Thomas W., and Darnell J.E. 1972. Evidence that all messenger RNA molecules (except histone
messenger RNA) contain poly(A) sequences and that the
poly(A) has a nuclear function. J. Mol. Biol. 71: 21.
Belsham G.J. and Jackson R.J. 2000. Translation initiation on picornavirus RNA. In Translational control of gene expression
(ed. N. Sonenberg et al.), p. 869. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Bergamini G., Preiss T., and Hentze M.W. 2000. Picornavirus
IRESes and the poly(A) tail jointly promote cap-independent
translation in a mammalian cell-free system. RNA 6: 1781.
Bi X. and Goss D.J. 2000. Wheat germ poly(A)-binding protein
increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F. J. Biol.
Chem. 275: 17740.
Blobel G. 1973. A protein of molecular weight 78,000 bound to
the polyadenylate region of eukaryotic messenger RNAs.
Proc. Natl. Acad. Sci. 70: 924.
Burd C.G., Matunis E.L., and Dreyfuss G. 1991. The multiple
RNA-binding domains of the mRNA poly(A)-binding protein
have different RNA-binding activities. Mol. Cell. Biol. 11:
3419.
Bushell M., Wood W., Carpenter G., Pain V.M., Morley S.J.,
and Clemens M.J. 2001. Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with
the poly(A)-binding protein by caspase- and viral proteasemediated cleavages. J. Biol. Chem. 276: 23922.
Chen C.Y. and Shyu A.B. 1994. Selective degradation of earlyresponse-gene mRNAs: Functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol. 14: 8471.
Christensen A.K., Kahn L.E., and Bourne C.M. 1987. Circular
polysomes predominate on the rough endoplasmic reticulum
of somatotropes and mammotropes in the rat anterior pituitary. Am. J. Anat. 178: 1.
Craig A.W., Haghighat A., Yu A.T., and Sonenberg N. 1998. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392: 520.
Deo R.C., Sonenberg N., and Burley S.K. 2001. X-ray structure
of the human hyperplastic discs protein: An ortholog of the Cterminal domain of poly(A)-binding protein. Proc. Natl.
Acad. Sci. 98: 4414.
Deo R.C., Bonanno J.B., Sonenberg N., and Burley S.K. 1999.
Recognition of polyadenylate RNA by the poly(A)-binding
protein. Cell 98: 835.
Doel M.T. and Carey N.H. 1976. The translational capacity of
deadenylated ovalbumin messenger RNA. Cell 8: 51.
Gallie D.R. 1991. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev.
5: 2108.
Gingras A.-C., Raught B., and Sonenberg N. 1999. eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and
regulators of translation. Annu. Rev. Biochem. 68: 913.
———. 2001. Regulation of translation initiation by
FRAP/mTOR. Genes Dev. 15: 807.
Görlach M., Burd C.G., and Dreyfuss G. 1994. The mRNA
poly(A)-binding protein: Localization, abundance, and RNAbinding specificity. Exp. Cell Res. 211: 400.
Gray N.K., Coller J.M., Dickson K.S., and Wickens M. 2000.
Multiple portions of poly(A)-binding protein stimulate translation in vivo. EMBO J. 19: 4723.
Grosset C., Chen C.Y., Xu N., Sonenberg N., Jacquemin-Sablon
H., and Shyu A.B. 2000. A mechanism for translationally
coupled mRNA turnover: Interaction between the poly(A) tail
and a c-fos RNA coding determinant via a protein complex.
Cell 103: 29.
Grossi de Sa M.F., Standart N., Martins de Sa C., Akhayat O.,
Huesca M., and Scherrer K. 1988. The poly(A)-binding protein facilitates in vitro translation of poly(A)-rich mRNA.
Eur. J. Biochem. 176: 521.
299
Gueydan C., Droogmans L., Chalon P., Huez G., Caput D., and
Kruys V. 1999. Identification of TIAR as a protein binding to
the translational regulatory AU-rich element of tumor necrosis factor alpha mRNA. J. Biol. Chem. 274: 2322.
Hershey J.W.B. and Merrick W.C. 2000. Pathway and mechanism of initiation of protein synthesis. In Translational control
of gene expression (ed. N. Sonenberg et al.), p. 33. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York.
Hoshino S., Imai M., Kobayashi T., Uchida N., and Katada T.
1999. The eukaryotic polypeptide chain releasing factor
(eRF3/GSPT) carrying the translation termination signal to the
3´-poly(A) tail of mRNA. Direct association of eRF3/GSPT
with polyadenylate-binding protein. J. Biol. Chem. 274:
16677.
Iizuka N., Najita L., Franzusoff A., and Sarnow P. 1994. Cap-dependent and cap-independent translation by internal initiation
of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol. Cell. Biol. 14: 7322.
Imataka H., Gradi A., and Sonenberg N. 1998. A newly identified
N-terminal amino acid sequence of human eIF4G binds
poly(A)-binding protein and functions in poly(A)-dependent
translation. EMBO J. 17: 7480.
Ito T. and Lai M.M. 1999. An internal polypyrimidine-tract-binding protein-binding site in the hepatitis C virus RNA attenuates
translation, which is relieved by the 3´-untranslated sequence.
Virology 254: 288.
Jacobson A. 1996. Poly(A) metabolism and translation: The
closed-loop model. In Translational control (ed. J.W.B. Hershey et al.), p. 451. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York.
Jacobson A. and Favreau M. 1983. Possible involvement of
poly(A) in protein synthesis. Nucleic Acids Res. 11: 6353.
Kates J. 1971. Transcription of the vaccinia virus genome and the
occurrence of polyriboadenylic acid sequences in messenger
RNA. Cold Spring Harbor Symp. Quant. Biol. 35: 743. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.
Khaleghpour K., Svitkin Y.V., Craig A.W., DeMaria C.T., Deo
R.C., Burley S.K., and Sonenberg N. 2001a. Translational repression by a novel partner of human poly(A) binding protein,
Paip2. Mol. Cell. 7: 205.
Khaleghpour K., Kahvejian A., De Crescenzo G., Roy G., Svitkin
Y.V., Imataka H., O’Connor-McCourt M., and Sonenberg N.
2001b. Dual interactions of the translational repressor Paip2
with poly(A) binding protein. Mol. Cell. Biol. 21: 5200.
Kieft J.S., Zhou K., Jubin R., and Doudna J.A. 2001. Mechanism
of ribosome recruitment by hepatitis C IRES RNA. RNA 7:
194.
Kozlov G., Trempe J.F., Khaleghpour K., Kahvejian A., Ekiel I.,
and Gehring K. 2001. Structure and function of the C-terminal
PABC domain of human poly(A)-binding protein. Proc. Natl.
Acad. Sci. 98: 4409.
Kuhn U. and Pieler T. 1996. Xenopus poly(A) binding protein:
Functional domains in RNA binding and protein-protein interaction. J. Mol. Biol. 256: 20.
Le H., Tanguay R.L., Balasta M.L., Wei C.C., Browning K.S.,
Metz A.M., Goss D.J., and Gallie D.R. 1997. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)binding protein and increase its RNA binding activity. J. Biol.
Chem. 272: 16247.
Lomakin I.B., Hellen C.U., and Pestova T.V. 2000. Physical association of eukaryotic initiation factor 4G (eIF4G) with
eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol. Cell. Biol. 20:
6019.
Lorenzetti D., Bohlega S., and Zoghbi H.Y. 1997. The expansion
of the CAG repeat in ataxin-2 is a frequent cause of autosomal
dominant spinocerebellar ataxia. Neurology. 49: 1009.
Mathews M.B., Sonenberg N., and Hershey J.W.B. 2000. Origins
and principles of translational control. In Translational control
of gene expression (ed. N. Sonenberg et al.), p. 1. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York.
Matsuda S., Kawamura-Tsuzuku J., Ohsugi M., Yoshida M., Emi
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
300
KAHVEJIAN, ROY, AND SONENBERG
M., Nakamura Y., Onda M., Yoshida Y., Nishiyama A., and
Yamamoto T. 1996. Tob, a novel protein that interacts with
p185erbB2, is associated with anti-proliferative activity.
Oncogene 12: 705.
Mazumder B., Seshadri V., Imataka H., Sonenberg N., and Fox
P.L. 2001. Translational silencing of ceruloplasmin requires
the essential elements of mRNA circularization: Poly(A) tail,
poly(A)-binding protein, and eukaryotic translation initiation
factor 4G. Mol. Cell. Biol. 21: 6440.
Munroe D. and Jacobson A. 1990a. Tales of poly(A): A review.
Gene 91: 151.
———. 1990b. mRNA poly(A) tail, a 3´ enhancer of translational initiation. Mol. Cell. Biol. 10: 3441.
Nagai K. 1996. RNA-protein complexes. Curr. Opin. Struct.
Biol. 6: 53.
Ostareck D.H., Ostareck-Lederer A., Shatsky I.N., and Hentze
M.W. 2001. Lipoxygenase mRNA silencing in erythroid differentiation: The 3´UTR regulatory complex controls 60S ribosomal subunit joining. Cell. 104: 281.
Ostareck D.H., Ostareck-Lederer A., Wilm M., Thiele B.J., Mann
M., and Hentze M.W. 1997. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3´ end. Cell. 89: 597.
Otero L.J., Ashe M.P., and Sachs A.B. 1999. The yeast poly(A)binding protein Pab1p stimulates in vitro poly(A)-dependent
and cap-dependent translation by distinct mechanisms. EMBO
J. 18: 3153.
Oubridge C., Ito N., Evans P.R., Teo C.H., and Nagai K. 1994.
Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an
RNA hairpin. Nature 372: 432.
Palatnik C.M., Wilkins C., and Jacobson A. 1984. Translational
control during early Dictyostelium development: Possible involvement of poly(A) sequences. Cell 36: 1017.
Perez-Canadillas J.M. and Varani G. 2001. Recent advances in
RNA-protein recognition. Curr. Opin. Struct. Biol. 11: 53.
Pestova T.V., Shatsky I.N., Fletcher S.P., Jackson R.J., and
Hellen C.U. 1998. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine
fever virus RNAs. Genes Dev. 12: 67.
Piecyk M., Wax S., Beck A.R., Kedersha N., Gupta M., Maritim
B., Chen S., Gueydan C., Kruys V., Streuli M., and Anderson
P. 2000. TIA-1 is a translational silencer that selectively regulates the expression of TNF-alpha. EMBO J. 19: 4154.
Piron M., Vende P., Cohen J., and Poncet D. 1998. Rotavirus
RNA-binding protein NSP3 interacts with eIF4GI and evicts
the poly(A) binding protein from eIF4F. EMBO J. 17: 5811.
Poncet D., Aponte C., and Cohen J. 1993. Rotavirus protein
NSP3 (NS34) is bound to the 3´ end consensus sequence of viral mRNAs in infected cells. J. Virol. 67: 3159.
———. 1996. Structure and function of rotavirus nonstructural
protein NSP3. Arch. Virol. Suppl. 12: 29.
Pyronnet S., Imataka H., Gingras A.-C., Fukunaga R., Hunter T.,
and Sonenberg N. 1999. Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E.
EMBO J. 18: 270.
Raught B., Gingras A.-C., and Sonenberg N. 2000. Regulation of
ribosomal recruitment in eukaryotes. In Translational control
of gene expression (ed. N. Sonenberg et al.), p. 245. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.
Sachs A.B. and Davis R.W. 1989. The poly(A) binding protein is
required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 58: 857.
———. 1990. Translation initiation and ribosomal biogenesis:
Involvement of a putative rRNA helicase and RPL46. Science
247: 1077.
Sachs A.B., Davis R.W., and Kornberg R.D. 1987. A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7:
3268.
Searfoss A., Dever T.E., and Wickner R. 2001. Linking the 3´
poly(A) tail to the subunit joining step of translation initiation:
Relations of pab1p, eukaryotic translation initiation factor 5b
(Fun12p), and Ski2p-Slh1p. Mol. Cell. Biol. 21: 4900.
Shibata H., Huynh D.P., and Pulst S.M. 2000. A novel protein
with RNA-binding motifs interacts with ataxin-2. Hum. Mol.
Genet. 9: 1303.
Stoecklin G., Hahn S., and Moroni C. 1994. Functional hierarchy
of AUUUA motifs in mediating rapid interleukin-3 mRNA decay. J. Biol. Chem. 269: 28591.
Tarun S.Z. and Sachs A.B. 1995. A common function for mRNA
5´ and 3´ ends in translation initiation in yeast. Genes Dev. 9:
2997.
———. 1996. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 15:
7168.
Tarun S.Z., Jr., Wells S.E., Deardorff J.A., and Sachs A.B. 1997.
Translation initiation factor eIF4G mediates in vitro poly(A)
tail-dependent translation. Proc. Natl. Acad. Sci. 94: 9046.
Tucker M., Valencia-Sanchez M.A., Staples R.R., Chen J., Denis
C.L., and Parker R. 2001. The transcription factor associated
Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell.
104: 377.
Varani G. and Nagai K. 1998. RNA recognition by RNP proteins
during RNA processing. Annu. Rev. Biophys. Biomol. Struct.
27: 407.
Vende P., Piron M., Castagne N., and Poncet D. 2000. Efficient
translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor
eIF4G and the mRNA 3´ end. J. Virol. 74: 7064.
Wakiyama M., Imataka H., and Sonenberg N. 2000. Interaction of
eIF4G with poly(A)-binding protein stimulates translation and
is critical for Xenopus oocyte maturation. Curr. Biol. 10: 1147.
Wei C.C., Balasta M.L., Ren J., and Goss D.J. 1998. Wheat germ
poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogues. Biochemistry 37: 1910.
Wells S.E., Hillner P.E., Vale R.D., and Sachs A.B. 1998. Circularization of mRNA by eukaryotic translation initiation factors.
Mol. Cell. 2: 135.
Wickens M., Goodwin E.B., Kimble J., Strickland S., and Hentze
M.W. 2000. Translational control of developmental decisions.
In Translational control of gene expression (ed. N. Sonenberg
et al.), p. 295. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York.
Widner W.R. and Wickner R.B. 1993. Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking
expression of viral mRNA. Mol. Cell. Biol. 13: 4331.
Xu N., Chen C.Y., and Shyu A.B. 1997. Modulation of the fate of
cytoplasmic mRNA by AU-rich elements: Key sequence features controlling mRNA deadenylation and decay. Mol. Cell.
Biol. 17: 4611.
Yoshida Y., Hosoda E., Nakamura T., and Yamamoto T. 2001.
Association of ANA, a member of the antiproliferative Tob
family proteins, with a caf1 component of the ccr4 transcriptional regulatory complex. Jpn. J. Cancer Res. 92: 592.
Yoshida Y., Matsuda S., Ikematsu N., Kawamura-Tsuzuku J., Inazawa J., Umemori H., and Yamamoto T. 1998. ANA, a novel
member of Tob/BTG1 family, is expressed in the ventricular
zone of the developing central nervous system. Oncogene 16:
2687.
Downloaded from symposium.cshlp.org on February 20, 2016 - Published by Cold Spring Harbor Laboratory Press
The mRNA Closed-loop Model: The Function of PABP and
PABP-interacting Proteins in mRNA Translation
A. KAHVEJIAN, G. ROY and N. SONENBERG
Cold Spring Harb Symp Quant Biol 2001 66: 293-300
Access the most recent version at doi:10.1101/sqb.2001.66.293
References
This article cites 71 articles, 40 of which can be accessed free at:
http://symposium.cshlp.org/content/66/293.refs.html
Article cited in:
http://symposium.cshlp.org/content/66/293#related-urls
Email alerting
service
Receive free email alerts when new articles cite this article - sign up in the
box at the top right corner of the article or click here
To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to:
http://symposium.cshlp.org/subscriptions
Copyright © 2001 Cold Spring Harbor Laboratory Press