Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA

Abstract

Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence have revealed that the full picture is more complex, and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA.

The crosstalk between transcription, translation and degradation of mRNA can be deduced intuitively for prokaryotes, since transcription and translation occur in the same time and space. Eukaryotes, on the other hand, segregate their chromosomes from the rest of the cell and the barrier of the nuclear membrane divides transcription from protein expression. It is now well established that transcription and nuclear mRNA processing are coupled, as mRNA maturation (capping, splicing and polyadenylation) and mRNA export are initiated on nascent transcripts.1 Even if a process like spliceosome assembly can be initiated during transcription, evidence suggest that some genes are spliced cotranscriptionally, while for other genes splicing occurs post-transcriptionally.2,3 Moreover, these events depend on interactions between the transcription machinery and RNA processing factors, in which the transcription elongation complex acts as a platform to recruit these factors.4 It is known that several RNA-binding proteins which specify the cytoplasmic fate of transcripts (i.e., translational control, mRNA decay or mRNA localization) shuttle between the cytoplasm and the nucleus, and their presence in the nucleus is essential for their function.5 For instance, the exon-junction complex (E°C), which is deposited on mRNA after splicing, follows transcripts to the cytoplasm, where it enhances mRNA translation, targets transcripts to nonsense-mediated decay (NMD) and participates in mRNA localization.5 However, it is still unclear to what extent these factors are recruited during transcription of their target mRNA, and how this occurs. Here, we review current evidence showing how the RNA polymerase II transcription machinery directly affects mRNA translation, localization or degradation in the cytoplasm by promoting the cotranscriptional recruitment of RNA-binding proteins involved in these processes. This evidence comes mostly from the budding yeast, in which these pathways have been well studied. However, since several of the factors involved are highly conserved throughout evolution, these mechanisms are likely relevant to higher eukaryotes as well.

Rpb4/7 Couples Transcription and Cytoplasmic mRNA Degradation

RNA polymerase II is constituted of 12 subunits in eukaryotes, including ten core subunits, which encompass the catalytic site, and the Rbp4-Rbp7 subcomplex (Rpb4/7), which forms a stalk-like protrusion on the RNA polymerase II core, in close proximity to the RNA exit channel.6 Rpb7 is known to bind the nascent RNA, although with little specificity.6 In yeast, deletion of RPB7 is lethal, while Rpb4 is essential for viability only at high temperature.7 Unlike RNA polymerase II core subunits, Rpb4 and Rbp7 are essential only for transcription initiation in vitro, and are dispensable for transcription elongation.8 Genome wide chromatin immunoprecipitation in yeast revealed that Rpb4/7 occupancy is identical to Rpb3, suggesting that this heterodimer is associated with RNA polymerase II throughout the transcription cycle.9 Like most small subunits of RNA polymerase II in yeasts (S. cerevisiae and S. pombe), Rpb4 is in excess compared to the large subunits Rpb1, Rpb2 and Rpb3.10,11 Moreover, Rpb4 and Rpb7 (and Rpb3 in humans cells12) are the only subunits that actively shuttle between the nucleus and the cytoplasm,13 suggesting other roles for Rpb4/7 beside transcription.

Indeed, Rpb4/7 has been found to affect the cytoplasmic degradation of mRNA in yeast. In the cytoplasm, Rpb4 enhances the degradation of specific transcripts, called protein biosynthetic factors mRNA (PBF mRNA) as they encode for ribosomal proteins or translational factors.14 The turnover of PBF mRNA is extremely sensitive to environmental stimuli, and Rpb4 regulates their deadenylation and degradation rate in different conditions. Rpb4 interacts physically and genetically with components of the major 5′ to 3′ mRNA degradation pathway in yeast, like Pat1 and Lsm2, and accumulates in cytoplasmic bodies called P bodies, which are sites of mRNA storage and degradation.15 A role for Rpb7 in cytoplasmic mRNA decay has also been demonstrated.16 The generation of thermosensitive mutants of Rpb7 in yeast revealed that some mutants displayed a defect in mRNA decay, and not in transcription, at the non-permissive temperature. These Rpb7 mutants compromised the degradation of both PBF and non-PBF transcripts, suggesting a more general role for Rpb7 in mRNA decay, compared to Rbp4. Unlike Rpb4, Rpb7 acts via both 5′ to 3′ and 3′ to 5′ mRNA degradation pathways, as it interacts physically and genetically with both pathways. While these results suggested a function for both Rpb4 and Rpb7 in cytoplasmic mRNA decay, it was still unclear if this function depends on the association of Rpb4/7 with the core subunits of RNA polymerase II.

Evidence emerged on the implication of the transcription machinery in Rpb4/7-dependent cytoplasmic mRNA decay.17 Specific thermosensitive mutants of RNA polymerase II (Rpb6^Q100R and Rpb1^{C67S, C70S}), which disrupt interactions with the Rpb4/7 heterodimer, display defect in mRNA degradation similar to those observed previously for Rpb4 and Rpb7 mutants. This phenotype is specific for these rpb6 and rpb1 alleles, since other alleles of RPB1 do not display a defective mRNA decay. Overexpression of Rpb4/7 partially rescues the defect in mRNA decay observed in the Rpb6^Q100R mutant, suggesting that the interaction between Rpb4/7 and RNA polymerase II is important for the role of Rpb4/7 in cytoplasmic mRNA degradation. These results strongly suggest that the cotranscriptional recruitment of Rpb4/7 by RNA polymerase II is necessary for its role in cytoplasmic mRNA decay, arguing against the possibility that the Rpb4/7 heterodimer can be recruited to mRNA independently of the RNA polymerase. While the role of Rpb4/7 in mRNA decay has been observed mostly with transcripts that are part of the PBF family, little is known about its role in the general degradation of mRNA. It would be interesting to investigate if this mechanism applies to all mRNA.

Mechanistic Coupling Between Transcription and Translation

Besides its role in cytoplasmic mRNA decay, a recent publication revealed another function for the Rpb4/7 complex in promoting efficient translation initiation.18 Indeed, yeast Rpb4/7 was found to physically interact with Nip1 and Hcr1, two components of the translation initiation factor 3 (eIF3), which serves as a platform for the recruitment of the small ribosomal subunit during translation initiation.19 Deletion of RPB4 and a conditional mutant allele of RPB7 (rpb7–26) show reduced formation of polysomes and hypersensitivity to translation inhibitors. Importantly, the rpb7–26 allele does not display any major defects in transcription or mRNA decay, suggesting that the effects observed on translation are not indirect. The role of Rpb4/7 in translation initiation was particularly evaluated during the stimulation of translation following exit from glucose starvation. During glucose starvation, mRNA is excluded from polysomes and stored in P bodies. When glucose is added back to yeast culture, the mRNA move from P bodies to the polysomes and resume translation.20 This process was significantly reduced in the rpb7–26 mutant and RPB4 deletion strains, demonstrating an implication of Rpb4/7 in translation initiation.

An important point in this study was that the role of Rpb4/7 in translation initiation depends on its interaction with the core RNA polymerase II. Indeed, mutants defective in interaction of RNA pol II with Rpb4/7 (Rpb6^Q100R and Rpb1^{C67S, C70S}) also exhibit abnormal pattern of translation. Furthermore, nuclear import of Rpb4 is essential to this new function, since a NLS-mutated Rpb4 displayed abnormal polysome profile. From these results, a model can be proposed in which RNA polymerase II regulates translation initiation by stimulating the binding of Rpb4/7 on nascent mRNA in the nucleus, before they reach the ribosomes and initiate translation in the cytoplasm.

Based on this study and on previous ones revealing a role in mRNA degradation, the Rpb4/7 heterodimer has been coined as an “mRNA coordinator” as this complex interacts with mRNA at each step of their life cycle—from transcription, translation to mRNA decay (Fig. 1). However, these results raise questions concerning the function of this coordination between the transcription machinery, translation and mRNA decay. A key observation is that the role of Rpb4/7 in these processes is particularly important during environmental stress conditions, like heat stress and glucose starvation.18 Indeed, while Rpb7 is an essential gene, Rpb4 is essential for transcription during temperature stress.21 Furthermore, Rpb4 is known to mediate nuclear export of mRNA during stress conditions and most Rpb4 molecules accumulate in the cytoplasm during stress.22 Altogether, these data suggest a role for Rpb4/7 in integrating cellular response to environmental stress by acting on mRNA, from their synthesis in the nucleus to their translation and decay in the cytoplasm.

Spt4-Spt5 Link Transcription to Cytoplasmic mRNA Localization

Beside translation and mRNA decay, recent evidence point toward a novel role for the transcription machinery in promoting cytoplasmic mRNA localization. mRNA localization is a mechanism which restricts spatial and temporal mRNA translation in a cell. This process is essential for the establishment of cellular polarity, synaptic plasticity, asymmetric cell division and cell fate determination.23 Evidence from several organisms suggests that the mRNA localization process is initiated on nascent mRNAs. For instance, the RNA-binding proteins ZBP1 and ZBP2, which are involved in β-actin mRNA localization at the leading edge in fibroblasts, are recruited on nascent β-actin mRNA.24,25 Localization of oskar mRNA at the posterior pole of the Drosophila embryo requires members of the exon junction complex (EJC), like Y14-Mago and eIFIIIA.26,27 While the assembly of the EJC takes place concomitantly with splicing and can occur cotranscriptionally, at least in mammalian cells,28 it is not clear yet if EJC assembly occurs cotranscriptionally in Drosophila. In both cases, it is still unclear if this recruitment is coupled to the transcription machinery, as it was shown for nuclear pre-mRNA processing pathways.

A link between transcription and mRNA localization has been more clearly established in the budding yeast, in which over 30 transcripts are actively transported and localized to the bud tip.29 mRNA localization to the bud depends on the RNA-binding protein She2, which forms with She3 and Myo4 a complex called the “locasome” that carries their cargo along the actin filaments to the bud tip.30 She2 recognizes specific RNA motifs, called localization elements or zipcodes, present within the coding sequence or 3′ untranslated region of localized transcripts.31 While it was already known that She2 associates with mRNA in the nucleus,32 recent evidence have made clear that She2 is actively imported in the nucleus via a non-classical nuclear localization signal (NLS), and that disrupting nuclear import of She2 (by mutating its NLS or retaining it in the cytoplasm) perturb mRNA localization at the bud and asymmetric protein synthesis.33,34

A role for the transcription machinery in the cotranscriptional recruitment of She2 to nascent bud-localized transcripts has been recently revealed.35 Using chromatin immunoprecipitation (ChIP), She2 was found to be associated with genes coding for bud-localized mRNA, in a transcription-dependent manner. Treatment with RNase or a mutation in the RNA-binding domain of She2 reduces its association with these genes, suggesting that She2 binds the nascent transcripts. Interestingly, She2 interacts in vivo with the elongating form of RNA polymerase II, raising the possibility that She2 may be recruited to chromatin via the transcription machinery. Indeed, She2 directly interacts with the transcription factor Spt4–Spt5, also known as DSIF, which is part of the transcription elongation complex.36 Spt4 and Spt5 form an heterodimeric complex which regulates the processivity of RNA pol II.37 Beside its role in elongation, Spt5 is also implicated in mRNA capping, as it interacts and activates the cap guanylyltransferase,38 and in mRNA splicing.39 Mutations in either SPT4 or SPT5 reduce the cotranscriptional recruitment of She2 on a gene coding for a bud-localized mRNA and disrupt its localization at the bud. This leads to a model in which Spt4–Spt5 serves as a “launching pad,” which favors the cotranscriptional loading of She2 on nascent transcripts (Fig. 2).

It is well understood that the cotranscriptional recruitment of mRNA processing factors imposes a temporal order on the different steps of pre-mRNA maturation (from 5′ end capping to mRNA export) and mediates quality control of transcripts before nuclear export.4 In comparison, it is not yet clear what the advantages for the cotranscriptional loading of RNA-binding proteins regulating cytoplasmic events, like translation, mRNA localization or decay, could be. One possibility is that interactions with the transcription machinery may ensure that RNA-binding factors specifying the cytoplasmic fate of an mRNA are loaded before mRNA export. Evidence from yeast, Drosophila and vertebrate show that, in the absence of their mRNA localization factors or translational repressors, localized mRNA are still exported from the nucleus.26,34,40 This suggests the absence of a nuclear quality control that would retain or degrade improperly loaded transcripts before their export to the cytoplasm, which would cause their premature translation, degradation or mislocalization. The cotranscriptional recruitment of factors which regulate the cytoplasmic fate of mRNA may help avoid this problem by loading these factors prior transcription termination.

Another possibility is that cotranscriptional recruitment allows an integration of different regulatory mechanisms before nuclear export of an mRNA. Rpb4/7 is a good example, as it regulates both mRNA translation and decay. It is also known that the presence of She2 in the nucleus is essential for the recruitment of the translational repressor Puf6 on the bud-localized ASH1 mRNA in yeast.34 Cotranscriptional recruitment of She2 on nascent mRNA may favor the subsequent loading of Puf6, and ensure that translational control of bud-localized transcripts is established before their export to the cytoplasm. It will be of interest to determine if other pathways mediating the cytoplasmic fate of mRNA are also coupled with transcription, and to what extent the transcription machinery primes mRNA for translational regulation, localization or decay in the cytoplasm.

Figures and Tables

Figure 1 Rpb4/7 is involved in transcription, translation and mRNA decay. Rpb4/7 associates with the core RNA polymerase II (pol II) subunits during elongation and is loaded cotranscriptionally on specific transcripts. In the cytoplasm, Rpb4/7 recruits eIF3 and promotes translation initiation. During stress, Rpb4/7 follows mRNA to P bodies and favors the return of transcripts to polysomes after stress. In addition, Rpb4/7 can promote the degradation of its target mRNA, via polyA tail shortening, followed by exonuclease degradation. As Rpb4/7 actively shuttles between the nucleus and cytoplasm, it can be re-imported to the nucleus.

Figure 2 Spt4–Spt5 promote the cotranscriptional recruitment of She2 on the nascent ASH1 mRNA. Interaction between She2 and Spt4–Spt5 on elongating RNA polymerase II (pol II) favors the transfer of She2 on localization elements RNA as they emerge from the polymerase. The bud-localized ASH1 mRNA contains four localization elements, which fold as stem-loop structures.

Acknowledgements

We thank Emmanuelle Querido for critical reading of the manuscript. This work was supported by the grant MOP43855 from the Canadian Institutes for Health Research (CIHR) to P.C. P.C is a Senior Scholar from the Fonds de la Recherche en Santé du Québec (FRSQ).