Subcellular localization of mRNA and factors ... - Semantic Scholar

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Biochemical Society Transactions (2008) Volume 36, part 4

Subcellular localization of mRNA and factors involved in translation initiation Nathaniel P. Hoyle and Mark P. Ashe1 Faculty of Life Sciences, The Michael Smith Building, The University of Manchester, Oxford Road, Manchester M13 9PT, U.K.

Abstract Both the process and synthesis of factors required for protein synthesis (or translation) account for a large proportion of cellular activity. In eukaryotes, the most complex and highly regulated phase of protein synthesis is that of initiation. For instance, across eukaryotes, at least 12 factors containing 22 or more proteins are involved, and there are several regulated steps. Recently, the localization of mRNA and factors involved in translation has received increased attention. The present review provides a general background to the subcellular localization of mRNA and translation initiation factors, and focuses on the potential functions of localized translation initiation factors. That is, as genuine sites for translation initiation, as repositories for factors and mRNA, and as sites of regulation.

Translation factor localization In eukaryotic cells, the broad restriction of genetic material and the protein synthetic machinery to the nucleus and cytoplasm respectively affords cells the opportunity to regulate gene expression via subcellular localization of proteins and RNA. Hence, a whole raft of transcription factors are excluded from their nuclear site of action and can be induced to enter the nucleus by external stimuli [1]. However, the regulation of gene expression via localization is not confined to the regulated entry of transcription factors to the nucleus. A variety of subcellular bodies and granules in both the nucleus and cytoplasm also play roles. For instance, splicing factors are regulated via their localization to nuclear speckles [2] and mRNA decay factors are localized to cytoplasmic sites of mRNA decay called Pbodies (processing bodies) [3]. In addition, in many different biological systems, mRNAs are specifically localized to bring about localized sources of particular proteins [4]. Prominent examples of this type of regulation have been identified in animal oocytes, early embryos and neuronal cells. The question of whether factors involved in protein synthesis are regulated at the level of localization has received much less attention. Nevertheless, translation factors, mRNAs and ribosomes can be enriched in regions where translation is favoured, such as around the endoplasmic reticulum [5,6], at the leading edge of migrating fibroblasts and at neuronal synapses [4]. Additionally, translation factors can accumulate in large cytoplasmic foci that may play a role in aiding regulation or improving the efficiency of translation initiation [7]. Finally, translation initiation factors are also known to accumulate in repositories during times of Key words: EGP-body, eukaryotic initiation factor (eIF), mRNA, processing body (P-body), stress granule, translation initiation. Abbreviations used: eEF, eukaryotic elongation factor; eIF, eukaryotic initiation factor; eIF2-Pi , phosphorylated eIF2; GEF, guanine-nucleotide-exchange factor; eRF, eukaryotic release factor; P-body, processing body; TIA-1, T-cell-restricted intracellular antigen 1. 1 To whom correspondence should be addressed (email [email protected]).

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translational challenge to the cell, which may alter the persistence of transcripts during cellular stress [8].

The mechanism of translation initiation During the initiation of protein synthesis, the mRNA 5 -cap and 3 -poly(A) tail are recognized by eIF (eukaryotic initiation factor) 4E and Pab1 respectively. Both factors interact with eIF4G, which facilitates communication between the mRNA ends in a closed loop [9]. The small ribosomal subunit also interacts with a host of specific translation initiation factors. eIF2, in its GTP-bound form, interacts with the methionyl initiator tRNA to form a ternary complex and, along with eIF3, eIF5 and eIF1, associates with the small ribosomal subunit, possibly as a large preformed complex called the MFC (multi-factor complex). The protein-decorated mRNA and ribosome complexes interact with one another to form the 48S pre-initiation complex (Figure 1). The mRNA 5 -untranslated region is then scanned in a 5 → 3 direction until an AUG initiation codon sequence is encountered. The AUG initiation codon recognition triggers hydrolysis of the GTP bound to eIF2 and subsequent release of eIF2–GDP. Following GTP hydrolysis, the large ribosomal subunit is recruited and protein synthesis proceeds to the elongation phase. The eIF2–GDP by-product of translation initiation is recycled by the pentameric GEF (guanine-nucleotide-exchange factor) eIF2B [10]. Translation elongation is the process by which the mRNA is decoded into a growing polypeptide chain. Amino-acyl tRNAs in complex with GTP-bound eEF (eukaryotic elongation factor) 1A interact with the translating ribosome by virtue of the anticodon–codon base pairing between the tRNA and the mRNA respectively. During elongation, eEF1A– GTP becomes hydrolysed and eEF1A–GDP is released from the translating complex. eEF1A–GDP is recycled analogously to eIF2–GDP by action of a GEF, in this case eEF1B. eEF2 is involved in the translocation of the ribosome along the mRNA after peptide bond formation. When a Biochem. Soc. Trans. (2008) 36, 648–652; doi:10.1042/BST0360648

Post-Transcriptional Control

Figure 1 Translation factors are localized in yeast During translation initiation, guanine nucleotide exchange occurs on eIF2, catalysed by the GEF eIF2B. In S. cerevisiae, the essential process of eIF2 recycling occurs within the cytoplasmic guanine-nucleotide-exchange body, which consists of at least eIF2 and eIF2B. During translational inhibition, mRNA recruitment to the 43S pre-initiation

lects the high local rate of translation [18]. In yeast, surveys of translation factor localization by C-terminal GFP (green fluorescent protein) fusion have revealed that, in general, the translational machinery is largely diffuse within the cytoplasm, with the notable exceptions of eIF2 and eIF2B [7,19].

complex is compromised and transcripts enter into cytoplasmic stress-dependent mRNA granules such as P-bodies or EGP-bodies.

The guanine-nucleotide-exchange body: a site of catalysis or regulation?

ribosome reaches a stop codon, the process of translation is terminated by the action of eRF (eukaryotic release factor) 1 and eRF3 [10].

Localized translation Localized translation is an important and energy-efficient means of accumulating protein at a specific site. Here, mRNAs associate with the cytoskeleton and are trafficked to their site of translation. In general, the mRNA is translationally repressed until this site is reached. Examples of this mode of localization can be found in oligodendrocytes, where mRNAs such as myelin basic protein are transported in granules to the dendritic synapse, in fibroblasts, where βactin mRNA is localized to the leading edge of the cell during migration, and in Drosophila embryos, where mRNAs such as bicoid are trafficked to the appropriate pole [4]. Several studies have identified a more direct association of translation with the cytoskeleton. Lenk et al. [11] showed that actively translating polyribosomes are associated with the cytoskeleton; furthermore, disruption of the actin cytoskeleton is known to inhibit protein synthesis [12,13]. eEF1A, eEF2 and eRF3 have also been shown to have numerous interactions with both the actin and tubulin components of the cytoskeleton in a variety of eukaryotes [14–17]. In addition, eIF4E, eIF4G and Pab1 localize to the perinuclear region in mammalian fibroblasts, consistent with endoplasmic reticulum association [5]. As both soluble proteins and proteins destined for secretion or membrane incorporation are synthesized at the endoplasmic reticulum, the increased density of translation factors probably ref-

Given that translation factors, polyadenylated mRNA and ribosomal components are diffuse within the cytoplasm in yeast, it is perhaps surprising to find that eIF2 and eIF2B are restricted to a single cytoplasmic focus [7,19]. The eIF2B body consists of a resident pool of eIF2B, that accounts for ∼40 % of the total eIF2B. It also harbours a pool of eIF2 that cycles through the body and accounts for ∼18 % of the total eIF2 [7]. The integrity of the eIF2B body is affected by the translational status of the cell. Inhibiting translation by inactivating the essential translation factor eIF3B, or adding the ribosome-targeting antibiotic cycloheximide, disrupts the body [7]. This is thought to be due to sequestration of eIF2 in stalled translation complexes and suggests that the integrity of the body is dependent upon a high rate of guanine nucleotide exchange occurring within the cell. Furthermore, there is no obvious proximal concentration of other translation factors or tRNA, suggesting that it is not a site of local translation or tRNA recruitment, pinpointing guanine nucleotide exchange as the likely function of the body (Figure 1). Consistent with this notion, stresses and mutations that lower eIF2B guanine-nucleotide-exchange activity reduce eIF2 flux through the eIF2B body [7]. The localization of guanine-binding proteins is a common theme throughout Eukaryota. Heterotrimeric GTP-binding proteins are bound to the plasma membrane where they are coupled to transmembrane receptors and play a crucial role in cellular signalling [20]. Monomeric GTP-binding proteins and their GEFs are more widely distributed throughout cells [21]. Correct localization of the GEF associated with each G-protein can be crucial to its function. For instance, Cdc42 (cell division cycle 42) protein requires that the GEF, frabin, can localize to actin [22]. Plasma membrane localization and activation of Ras family G-proteins can only be achieved when the GEF, Sos (Son of sevenless), is localized to the membrane [23]. The localization of eIF2 and its GEF, eIF2B, to a specific locale can therefore be seen as a common theme among GEFs, which, in some cases, is essential to their function [24]. The observation that the cellular level of eIF2B is far exceeded by eIF2 would seem to support a hypothesis where guanine nucleotide exchange would need to be concentrated for maximal rates of protein synthesis [25]. Another possibility is that the existence of the eIF2B body facilitates the regulation of guanine nucleotide exchange. It is striking that the two factors that are spatially restricted are also involved centrally in the regulation of translation initiation [26]. Kinases such as Gcn2p phosphorylate the α C The


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subunit of eIF2 in response to cellular stress, causing a rapid cessation of translation initiation [27]. Phosphorylated eIF2 (eIF2-Pi ) inhibits the guanine-nucleotide-exchange activity of eIF2B; the consequent reduction in cellular eIF2–GTP levels is sufficient to curtail translation rapidly and efficiently. Part of this efficiency is due to the increased affinity of eIF2-Pi for eIF2B, making it a highly competitive inhibitor [28]. This effect is detectable microscopically, observed as a GCN2dependent increase in the level of eIF2 held present in the eIF2B body from ∼18 % to ∼36 % [7]. The rate of eIF2 cycling through the guanine-nucleotide-exchange body, as measured by FRAP (fluorescence recovery after photobleaching), was severely curtailed by amino acid starvation induced phosphorylation of eIF2 in a GCN2-dependent manner [7]. It is plausible that the spatial restriction of eIF2 and eIF2B facilitates regulation of guanine nucleotide exchange by Gcn2p. Localizing both the inhibitor, eIF2-Pi , and substrate, eIF2B, to the same focus is likely to lead to an effective inhibition of eIF2B function. However, Gcn2p is thought to be present at the ribosome [29], and no evidence for Gcn2p localization to the eIF2B body has been found (S.G. Campbell and M.P. Ashe, unpublished work). Therefore it seems likely that eIF2 phosphorylation does not occur at the eIF2B body and that the increased eIF2-Pi present at the body after amino acid starvation reflects the normal flux of eIF2 to the eIF2B body.

Translational repositories During times of cellular stress, where translation is attenuated, translation initiation factors become localized to cytoplasmic granules that are variously thought of as sites of mRNA storage, triage or degradation [8]. Cytoplasmic P-bodies are the only cytoplasmic mRNA granules conserved throughout eukaryotes. P-bodies were originally described as accumulations of mRNA degradation factors in mice [30] and humans [31–33], before being characterized as sites of mRNA storage and decay in Saccharomyces cerevisiae [3]. P-bodies are dynamic structures that increase in abundance in response to cellular stresses such as glucose starvation, osmotic shock, UV irradiation and high culture density [19]. It is thought that the translational inhibition associated with these conditions causes an increase in the flux of mRNA into P-bodies, which is observed as an increase in their size and abundance [19,34]. Similarly, decreasing flux out of P-bodies, by mutating mRNA-degrading enzymes such as Dcp1p and Xrn1p, causes similar increases in P-bodies [3]. P-bodies have also been shown to contain certain translation initiation factors: eIF4E in higher eukaryotes and eIF4E, eIF4G and Pab1p in S.cerevisiae [35–38]. mRNA translation and degradation are antagonistic processes. Inactivation of translation initiation factors induces both decapping and deadenylation of mRNA [39]. Furthermore, tethering Pab1p to a transcript is sufficient to increase its stability in vivo [40]. The reverse is also true: deletion of degradation factors such as Dcp1p, Pat1p and Dhh1p affords the cell a translational resistance to glucose starvation [41,42]. A competition between eIF4E and the decapping factors for mRNA cap C The


structure probably explains the basis of these effects. As Pbodies contain both translation and degradation factors, it is therefore a site where this antagonism is likely to be relevant. Importantly, in yeast, the recruitment of eIF4E to P-bodies is kinetically distinct from the formation of the body [36,38]. It is possible that recruitment of translation factors to the P-body is indicative of mRNAs being prepared to re-enter translation. However, it is not known how, before recruitment of eIF4E, eIF4G and Pab1p to P-bodies, mRNA might retain its cap and poly(A) tail at sites of high decapping enzyme and deadenylase concentration. An alternative possibility is that the recruitment of closed-loop factors to P-bodies may be associated with the recruitment and incipient degradation of closed-loop transcripts. Removal of the closed-loop factors from mRNA would be necessary for degradation to occur, and the high concentration of degradation factors in P-bodies may stimulate this process [43]. Related species of cytoplasmic mRNA granules have been described in both yeast and higher eukaryotes [8,38]. In higher eukaryotes, stalled translation complexes known as stress granules are induced by phosphorylation of eIF2 [44]. The prion-like proteins TIA-1 (T-cell-restricted intracellular antigen 1) and TIAR (TIA-1-related protein) bind to the ribosomal pre-initiation complex in lieu of ternary complex during times of translational challenge and induce aggregation of 48S pre-initiation complex components, including small ribosomal subunits eIF3, eIF2, eIF4G, eIF4E and Pab1p, and a host of mRNA-binding proteins [44–46]. Stress granules share some components with P-bodies, notably eIF4E, but also a variety of translational suppressors [8]. An observed juxtaposition of stress granules and P-bodies has led to the hypothesis that mRNAs are passed from stress granules to P-bodies [47]. This would ascribe a triage function to stress granules wherein only those transcripts destined for destruction are trafficked into P-bodies. In S. cerevisiae, a granular species containing mRNA, eIF4E, eIF4G and Pab1p, known as EGP-bodies, has been identified [38]. Despite many compositional differences, EGP-bodies may be at least functionally homologous with stress granules (Figure 2). Like stress granules, EGP-bodies consist of non-translating pre-initiation complexes, in this case closed-loop mRNPs (messenger ribonucleoproteins) not associated with the ribosome [38]. EGP-bodies lack mRNA degradation components and, like stress granules, are highly concentrated with translation factors, suggesting that mRNA storage is likely to be favoured [38,45]. Stress granules and EGP-bodies form after P-bodies. In higher eukaryotes, P-bodies are constitutive and stress granules are induced by translational challenge [47]. In yeast, both P-bodies and EGP-bodies are induced by stress, but EGP-bodies form at least 20 min after P-bodies [38]. This raises the question of how storage bodies can form after degradation bodies. One possibility is that the formation of visible granules is secondary to the function of these granules. In the case of P-bodies, it has been shown that mRNA degradation can proceed unimpaired without visible P-bodies [48]. It is therefore possible that stalled closed-loop

Post-Transcriptional Control

Figure 2 Stress-dependent granules in yeast and mammalian systems (A) In yeast, mRNA recruitment to the ribosome is compromised by severe translational stress, causing transcripts to accumulate in EGP-bodies and P-bodies. The divergent composition of these granules implies storage and degradation roles respectively. (B) In higher eukaryotes, eIF2 phosphorylation causes TIA-1/TIAR (TIA-1-related protein) to be recruited to ribosomes in lieu of the ternary complex, which promotes aggregation of the 40S subunit, mRNA and early translation factors into stress granules. Transcripts in stress granules may be subject to triage and subsequently resituated into P-bodies.

complexes are stored as soon as translation halts and that aggregation is a much slower process. During the translational attenuation associated with glucose starvation in yeast, mRNA has been shown to enter both P-bodies and EGP-bodies [38]. The functional consequences of occupancy of these granules are as yet unknown. However, it is a possibility that mRNA populations can be differentially distributed between the two granules. Occupancy of a Pbody, where the concentration of decay factors is high, would be more likely to induce degradation of a transcript than occupancy of an EGP-body, where a high concentration of translation factors and lack of decapping factors would probably prescribe storage of mRNA. It is possible that the targeting of transcripts to different cytoplasmic mRNA granules may represent an additional means to exert control over gene expression by altering the persistence of transcripts during translational attenuation.

Conclusions Translation initiation and its regulation are complicated processes in eukaryotic cells. The potential for regulation at the level of subcellular localization adds another facet to the intricacy. Challenges for the future will include a more detailed analysis of both the mechanism by which factors

become localized and the composition of the bodies/granules to which they localize.

N.P.H. is supported by Wellcome Trust VIP (Value in People) funding. The work in M.P.A.’s laboratory is funded by grants from The Wellcome Trust and The Leverhulme Trust.

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Received 10 March 2008 doi:10.1042/BST0360648