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Gene expression and its regulation depend on mRNA degradation. In eukaryotes, degradation is controlled by deadenylation rates, since a short poly(A) tail is considered to be the signal that activates decapping and triggers mRNA degradation. In contrast to this view, we show that global stability of mRNAs can be explained by variations in decapping speed alone. Rapid decapping of unstable mRNAs, for example, allows little time for deadenylation, which explains their longer than average poly(A) tails. As predicted by modeling of RNA degradation kinetics, mRNA stabilization in the absence of decapping led to a decrease in the length of the poly(A) tail, while depletion of deadenylases only increased the tail length. Our results suggest that decapping activation dictates mRNA stability independent of the deadenylation speed.One-Sentence SummaryUnstable mRNAs are characterized by rapid 5’ cap removal, independent of a prior shortening of the poly(A) tail.

Regulation of cellular processes is of key importance for survival of cells. Many regulations are mediated by the CCR4-Not complex, a highly conserved protein complex, which is present in eucaryotic cells, from yeast to mammals. In this work I study mostly the role of the Ccr4 subunit on yeast survival in the aging colony of Saccharomyces cerevisiae, mainly during cell cycle progression. Ccr4 si a deadenylase, and its main function is cleavage of poly(A) tail of mRNA molecules, and by doing so, shortening the mRNA life-time. It is very important during strictly regulated cellular processes, because it is essential that gene expression of specific genes happens only at specific time. Saccharomyces cerevisiae BY 4742 is a haploid yeast strain, which can easily be used for making deletion mutants in specific genes. In this diploma thesis I focus on studying a deletion mutant of CCR4, along with deletion of other genes, which influence yeast cell cycle. By comparing the phenotype of these mutants with wild type, it is possible to identify changes in phenotype, caused by these deletions, and their influence on yeast cell survival. Key words: cell processes regulation, CCR4-Not complex, Ccr4 subunit, cell cycle, yeast, Saccharomyces cerevisiae, gene deletion

Poly(A) binding protein cytoplasmic 1 (PABPC1) is an essential translational initiation factor. PABPC1 recognizes proteins through conserved PABPC1-interacting motifs 1 and 2 (PAM1 and PAM2). PABPC1-interacting protein-2 (Paip2) interacts with PABPC1 and modulates its activities. Here, we report that the formation of Paip2/PABPC1 complex protects it from proteasome independent degradation. We also show that PAM2 is critical for Paip2/PABPC1 interaction in vivo, in agreement with the observation that Paip2 requires PAM2 to interact with PABPC1 on mRNA. Lastly, we propose a role for Paip2 in displacing PABPC1 at the final stage of mRNA deadenylation when the poly(A) tail is partly degraded.

In many cell types, the length of the poly(A) tail of an mRNA is closely linked to its fate - a long tail is associated with active translation, a short tail with silencing and degradation. During mammalian oocyte development, two contrasting patterns of polyadenylation have been identified. Some mRNAs carry a long poly(A) tail during the growth stage and are actively translated, then become deadenylated and down-regulated during the subsequent stage, termed meiotic maturation. Other mRNAs carry a short tail poly(A) tail and are translationally repressed during growth, and their poly(A) tail lengthens and they become translationally activated during maturation. As well, a program of elimination of this ‘maternal’ mRNA is initiated during oocyte maturation. Here we describe a third pattern of polyadenylation: mRNAs are deadenylated in growing oocytes, become polyadenylated during early maturation and then deadenylated during late maturation. We show that the deadenylase, CNOT6, is present in cortical foci of oocytes and regulates deadenylation of these mRNAs, and that PUF-binding elements (PBEs) regulate deadenylation in mature oocytes. Unexpectedly, maintaining a long poly(A) tail neither enhances translation nor inhibits degradation of these mRNAs. Our findings implicate multiple machineries, more complex than previously thought, in regulating mRNA activity in oocytes.

The earliest step in the mRNA degradation process is deadenylation, a progressive shortening of the mRNA poly(A) tail by deadenylases. The question of when deadenylation takes place remains open. MYC mRNA is one of the rare examples for which it was proposed a shortening of the poly(A) tail during ongoing translation. In this study, we analyzed the poly(A) tail length distribution of various mRNAs, including MYC mRNA. The mRNAs were isolated from the polysomal fractions of polysome profiling experiments and analyzed using ligase-mediated poly(A) test analysis. We show that, for all the mRNAs tested with the only exception of MYC, the poly(A) tail length distribution does not change in accordance with the number of ribosomes carried by the mRNA. Conversely, for MYC mRNA, we observed a poly(A) tail length decrease in the fractions containing the largest polysomes. Because the fractions with the highest number of ribosomes are also those for which translation termination is more frequent, we analyzed the poly(A) tail length distribution in polysomal fractions of cells depleted in translation termination factor eRF3. Our results show that the shortening of MYC mRNA poly(A) tail is alleviated by the silencing of translation termination factor eRF3. These findings suggest that MYC mRNA is co-translationally deadenylated and that the deadenylation process requires translation termination to proceed.

Involved with important cellular or gene functions and implicated with many kinds of cancers, piRNAs, or piwi-interacting RNAs, are of small non-coding RNA with around 19–33 nt in length. Given a small non-coding RNA molecule, can we predict whether it is of piRNA according to its sequence information alone? Furthermore, there are two types of piRNA: one has the function of instructing target mRNA deadenylation, and the other does not. Can we discriminate one from the other? With the avalanche of RNA sequences emerging in the postgenomic age, it is urgent to address the two problems for both basic research and drug development. Unfortunately, to the best of our knowledge, so far no computational methods whatsoever could be used to deal with the second problem, let alone deal with the two problems together. Here, by incorporating the physicochemical properties of nucleotides into the pseudo K-tuple nucleotide composition (PseKNC), we proposed a powerful predictor called 2L-piRNA. It is a two-layer ensemble classifier, in which the first layer is for identifying whether a query RNA molecule is piRNA or non-piRNA, and the second layer for identifying whether a piRNA is with or without the function of instructing target mRNA deadenylation. Rigorous cross-validations have indicated that the success rates achieved by the proposed predictor are quite high. For the convenience of most biologists and drug development scientists, the web server for 2L-piRNA has been established at http://bioinformatics.hitsz.edu.cn/2L-piRNA/, by which users can easily get their desired results without the need to go through the mathematical details.

Summary Poly(A) tail length and mRNA deadenylation play important roles in gene regulation. However, how they regulate embryonic development and pluripotent cell fate is not fully understood. Here we present evidence that CNOT3-dependent mRNA deadenylation governs the pluripotent state. We show that CNOT3, a component of the Ccr4-Not deadenylase complex, is required for mouse epiblast maintenance. It is highly expressed in blastocysts and its deletion leads to peri-implantation lethality. The epiblast cells in Cnot3 deletion embryos are quickly lost during diapause and fail to outgrow in culture. Mechanistically, CNOT3 C terminus is required for its interaction with the complex and its function in embryonic stem cells (ESCs). Furthermore, Cnot3 deletion results in increases in the poly(A) tail lengths, half-lives, and steady-state levels of differentiation gene mRNAs. The half-lives of CNOT3 target mRNAs are shorter in ESCs and become longer during normal differentiation. Together, we propose that CNOT3 maintains the pluripotent state by promoting differentiation gene mRNA deadenylation and degradation, and we identify poly(A) tail-length regulation as a post-transcriptional mechanism that controls pluripotency., Highlights • CNOT3 is required for mouse epiblast maintenance during early development • CNOT3 C-terminal domain is necessary for the maintenance of the pluripotent state • CNOT3 promotes differentiation gene mRNA deadenylation and degradation • mRNA poly(A) tail regulation plays a critical role in pluripotency, In this article, Hu and colleagues showed that CNOT3, a component of the Ccr4-Not deadenylase complex, is required for the maintenance of the pluripotent state in mouse epiblast and embryonic stem cells. They found that CNOT3 promotes differentiation gene mRNA deadenylation and degradation, and post-transcriptionally regulates the gene expression program in pluripotent cells.

SummaryFor all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rate constants, which correspond to cytoplasmic lifetimes. Indeed, with few exceptions, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from either endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rate constants of short-tailed mRNAs vary broadly (1000-fold) and are more rapid for short-tailed mRNAs that had previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rate constants to impart a similarly large range in stabilities.HighlightsmRNAs enter the cytoplasm with diverse intra- and intergenic lengthsmRNA deadenylation rates span a 1000-fold range and correspond to mRNA half-livesAfter their tails become short, mRNAs decay at rates that span a 1000-fold rangeMore rapidly deadenylated mRNAs decay more rapidly upon reaching short tail lengths

Recent models of miRNA-mediated translational repression favor initiation as the phase of translation, where the repression is primarily evoked. miRNA-induced silencing complex utilizes miRNA as a guide molecule to find its target mRNA. To inhibit mRNA translation and induce its decay, many other proteins are then recruited, e.g. GW182 protein, PAN2-PAN3 a CCR4-NOT deadenylation complexes, decapping enzyme and eIF4E-binding proteins. miRNA-mediated translational repression is then established by either their concerted action or their consecutive and coordinated binding to target mRNA. To date, several mechanisms have been described: GW182- mediated PABP displacement; GW182-independent translational repression; miRNA-mediated eIF4A1 dissociation or eIF4A2 recruitment; translational repression mediated by deadenylases CAF1 and CCR4; and 4E-T, eIF4E2, GIGYF2 translational repressor proteins recruitment. Here, I provide up-to-the-minute summarization of these mechanisms with the aim to extract any universality from them.

Summary Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>∼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the ∼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.

Autophagy plays a context-dependent role in cardiac homeostasis. In this issue of Science Signaling , Yamaguchi et al . delineate a role for CCR4-NOT–mediated mRNA deadenylation in preventing the autophagy factor Atg7 from coactivating p53-mediated transcription of cell death genes in the heart.

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.

Summary Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis . Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis . Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.

The mRNA deadenylation involves the deadenylation complexes PAN2-PAN3 and CCR4-NOT-TOB and the translation termination complex eRF1-eRF3. All three proteins, eRF3, PAN3 and TOB, interact with the PABP protein. However, the role of eRF3 is still unclear. It has been reported that eRF3, TOB and PAN3 compete for the binding to PABP. Recently, it has been suggested that eRF3 may regulate mRNA deadenylation in a translation termination-coupled manner. In human, the gene eRF3/GSPT1, contains a trinucleotide GGC repeat in its 5’ end which lead to 5 allelic forms of the gene. There are five known alleles of this gene (7, 9, 10, 11 and 12-GGC). A strong correlation between the longest allele (12-GGC) and gastric and breast cancer development has been reported. Our project was (i) to improve our understanding on the role of eRF3 in the coupling of mRNA deadenylation with translation termination, (ii) to understand whether the GGC repeat polymorphism of eRF3 influences eRF3-PABP interaction. The kinetic measurements of eRF3-PABP interaction obtained by Surface Plasmon Resonance (SPR) show that the affinity of the allelic 12-GGC form is 10 fold lower than that of eRF3a (10-GGC). This decrease is mostly due to difference in the association rate of the complex. The weaker affinity of the 12-GGC allelic form may result in a deregulation of deadenylation, at least for some mRNAs, and thus, could promote cell proliferation and carcinogenesis. In fine, we show that the N-terminal region of eRF3 containing the glycine expansion plays a key role in the eRF3-PABP interaction, in the deadenylation process, and hence, in mRNA stability., La désadénylation des ARNm fait intervenir les complexes de désadénylation PAN2-PAN3 et CCR4-NOT-TOB mais aussi le complexe de terminaison de la traduction eRF1-eRF3. Ces trois complexes ont la capacité d'interagir avec la protéine PABP. Cependant, le rôle d'eRF3 n'est pas clairement établi. Il a été décrit que les facteurs eRF3, PAN3 et TOB sont en compétition pour l'interaction avec PABP et qu'il y a un couplage entre la terminaison de la traduction et la désadénylation assuré par eRF3. Chez l'homme, le gène eRF3/GSPT1 présente 5 formes alléliques qui diffèrent par le nombre de répétitions de codons GGC à l'extrémité 5' du cadre de lecture (7, 9, 10, 11 et 12-GGC). Une corrélation entre l'allèle 12-GGC et le risque de développement de cancer du sein et de l'estomac a été mis en évidence. Notre objectif est (i) d'améliorer notre compréhension du rôle d'eRF3 dans le processus de couplage traduction-dégradation des ARNm, (ii) de comprendre l'effet du polymorphisme de la région N-terminale d'eRF3 sur son interaction avec PABP. A travers la méthode de résonnance plasmonique de surface (SPR), nous montrons que l'affinité de la forme allélique 12-GGC est 10 fois plus faible que celle d'eRF3a (10-GGC). Cette différence est essentiellement due à la plus faible association de la forme 12-GGC avec PABP. La plus faible affinité de la forme 12-GGC d'eRF3 entrainerait une dérégulation de la désadénylation au moins pour certains ARNm et pourrait ainsi promouvoir la prolifération cellulaire et la carcinogenèse. La région N-terminale d'eRF3 contenant la répétition de glycine joue un rôle crucial dans l'interaction eRF3-PABP, dans la désadénylation et donc dans la stabilité de l'ARNm.

The mRNA deadenylation involves the deadenylation complexes PAN2-PAN3 and CCR4-NOT-TOB and the translation termination complex eRF1-eRF3. All three proteins, eRF3, PAN3 and TOB, interact with the PABP protein. However, the role of eRF3 is still unclear. It has been reported that eRF3, TOB and PAN3 compete for the binding to PABP. Recently, it has been suggested that eRF3 may regulate mRNA deadenylation in a translation termination-coupled manner. In human, the gene eRF3/GSPT1, contains a trinucleotide GGC repeat in its 5’ end which lead to 5 allelic forms of the gene. There are five known alleles of this gene (7, 9, 10, 11 and 12-GGC). A strong correlation between the longest allele (12-GGC) and gastric and breast cancer development has been reported. Our project was (i) to improve our understanding on the role of eRF3 in the coupling of mRNA deadenylation with translation termination, (ii) to understand whether the GGC repeat polymorphism of eRF3 influences eRF3-PABP interaction. The kinetic measurements of eRF3-PABP interaction obtained by Surface Plasmon Resonance (SPR) show that the affinity of the allelic 12-GGC form is 10 fold lower than that of eRF3a (10-GGC). This decrease is mostly due to difference in the association rate of the complex. The weaker affinity of the 12-GGC allelic form may result in a deregulation of deadenylation, at least for some mRNAs, and thus, could promote cell proliferation and carcinogenesis. In fine, we show that the N-terminal region of eRF3 containing the glycine expansion plays a key role in the eRF3-PABP interaction, in the deadenylation process, and hence, in mRNA stability., La désadénylation des ARNm fait intervenir les complexes de désadénylation PAN2-PAN3 et CCR4-NOT-TOB mais aussi le complexe de terminaison de la traduction eRF1-eRF3. Ces trois complexes ont la capacité d'interagir avec la protéine PABP. Cependant, le rôle d'eRF3 n'est pas clairement établi. Il a été décrit que les facteurs eRF3, PAN3 et TOB sont en compétition pour l'interaction avec PABP et qu'il y a un couplage entre la terminaison de la traduction et la désadénylation assuré par eRF3. Chez l'homme, le gène eRF3/GSPT1 présente 5 formes alléliques qui diffèrent par le nombre de répétitions de codons GGC à l'extrémité 5' du cadre de lecture (7, 9, 10, 11 et 12-GGC). Une corrélation entre l'allèle 12-GGC et le risque de développement de cancer du sein et de l'estomac a été mis en évidence. Notre objectif est (i) d'améliorer notre compréhension du rôle d'eRF3 dans le processus de couplage traduction-dégradation des ARNm, (ii) de comprendre l'effet du polymorphisme de la région N-terminale d'eRF3 sur son interaction avec PABP. A travers la méthode de résonnance plasmonique de surface (SPR), nous montrons que l'affinité de la forme allélique 12-GGC est 10 fois plus faible que celle d'eRF3a (10-GGC). Cette différence est essentiellement due à la plus faible association de la forme 12-GGC avec PABP. La plus faible affinité de la forme 12-GGC d'eRF3 entrainerait une dérégulation de la désadénylation au moins pour certains ARNm et pourrait ainsi promouvoir la prolifération cellulaire et la carcinogenèse. La région N-terminale d'eRF3 contenant la répétition de glycine joue un rôle crucial dans l'interaction eRF3-PABP, dans la désadénylation et donc dans la stabilité de l'ARNm.

Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

Roquin proteins mediate mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs via their Roquin domain. Here we present the crystal structure of a Roquin domain, revealing a mostly helical protein fold bearing a winged helix-turn-helix motif. By combining structural, biochemical and mutation analyses, we gain insight into the mode of RNA binding. We show that the winged helix-turn-helix motif is involved in the binding of constitutive decay elements-containing stem-loop mRNAs. Moreover, we provide biochemical evidence that Roquin proteins are additionally able to bind to duplex RNA and have the potential to be functional in different oligomeric states.

Structural analyses reveal the asymmetric assembly of Neurospora crassa PAN2–PAN3 complex and, along with functional work on the proteins from different species, indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay. The PAN2–PAN3 complex functions in general and microRNA-mediated mRNA deadenylation. However, mechanistic insight into PAN2 and its complex with the asymmetric PAN3 dimer is lacking. Here, we describe crystal structures that show that Neurospora crassa PAN2 comprises two independent structural units: a C-terminal catalytic unit and an N-terminal assembly unit that engages in a bipartite interaction with PAN3 dimers. The catalytic unit contains the exonuclease domain in an intimate complex with a potentially modulatory ubiquitin-protease–like domain. The assembly unit contains a WD40 propeller connected to an adaptable linker. The propeller contacts the PAN3 C-terminal domain, whereas the linker reinforces the asymmetry of the PAN3 dimer and prevents the recruitment of a second PAN2 molecule. Functional data indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay, which lead to complete mRNA degradation.

Deadenylation is the removal of poly(A) tails from mRNA. This chapter presents two methods to assay deadenylation in vitro. The first is a quick and quantitative assay for the degradation of radiolabeled poly(A) that can easily be adapted to be used for many different enzymes. The second method uses an extract from Drosophila embryos to catalyze the deadenylation of an RNA dependent on a specific sequence that also directs deadenylation in vivo.

Control of mRNA turnover is an essential step in the regulation of gene expression in eukaryotes. The concerted action of many enzymes regulates the way each mRNA is degraded. Moreover, the degradation of each mRNA is influenced by the environment surrounding the cell. The connection between the environment and changes in the half-lives of mRNAs is regulated by the activity of stress activated MAP kinases (SAPKs) and their substrates. Here, we will describe some of those mechanisms, and how SAPKs regulate mRNA stability in eukaryotic cells.

Post-transcriptional mRNA regulation is often attained by lengthening or shortening the 3′ poly(A) tail of a transcript. Eukaryotic mRNAs show a spectrum of deadenylation rates, thus allowing intricate control of gene expression, but the mechanisms that determine such rates are unclear. Three new studies highlight the structural and biochemical features of a key enzyme in removing poly(A) tails, the PAN2–PAN3 complex, providing clues to how different mRNA deadenylation rates can be achieved.

MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3′-untranslated regions of target mRNAs. Here we describe a recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5′-cap and 3′-poly(A) tail, resulting in translational repression.

International audience; In mammalian cells, certain mRNAs encoding cytokines or proto-oncogenes are especially unstable, because of the presence of a particular sequence element in their 3'-untranslated region named ARE (A/U-rich element). AREs cause this instability by provoking the rapid shortening of the poly(A) tail of the mRNA. The deadenylation of mRNAs mediated by AREs containing repeats of the AUUUA motif (class I/II AREs) is conserved in Xenopus embryos. Here, we first extend these observations by showing that c-Jun ARE, a representative of class III (non-AUUUA) AREs, also provokes the deadenylation of a reporter RNA in Xenopus embryos. Next, by immunodepletion and immunoneutralization experiments, we show that, in Xenopus, the rapid deadenylation of RNAs that contain the c-Jun ARE, but not an AUUUA ARE, requires EDEN-BP. This RNA-binding protein was previously shown to provoke the rapid deadenylation of certain Xenopus maternal RNAs. Finally, we show that CUG-BP, the human homologue of EDEN-BP, specifically binds to c-Jun ARE. Together, these results identify CUG-BP as a plausible deadenylation factor responsible for the post-transcriptional control of c-Jun proto-oncogene mRNA in mammalian cells.

Eukaryotic messenger RNAs are degraded through a pathway that starts with the removal of the poly(A) tail. A recent study shows that cytoplasmic mRNA deadenylation in mammals occurs by the consecutive action of two distinct deadenylases and that degradation of nonsense mRNA involves the same biphasic deadenylation pathway.

Eukaryotic mRNA deadenylation is generally considered as a two-step process in which the PAN2-PAN3 complex initiates the poly(A) tail degradation while, in the second step, the CCR4-NOT complex completes deadenylation, leading to decapping and degradation of the mRNA body. However, the mechanism of the biphasic poly(A) tail deadenylation remains enigmatic in several points such as the timing of the switch between the two steps, the role of translation termination and the mRNAs population involved. Here, we have studied the deadenylation of endogenous mRNAs in human cells depleted in either PAN3 or translation termination factor eRF3. Among the mRNAs tested, we found that only the endogenous ATF4 mRNA meets the biphasic model for deadenylation and that eRF3 prevents the shortening of its poly(A) tail. For the other mRNAs, the poor effect of PAN3 depletion on their poly(A) tail shortening questions the mode of their deadenylation. It is possible that these mRNAs experience a single step deadenylation process. Alternatively, we propose that a very short initial deadenylation by PAN2-PAN3 is followed by a rapid transition to the second phase involving CCR4-NOT complex. These differences in the timing of the transition from one deadenylation step to the other could explain the difficulties encountered in the generalization of the biphasic deadenylation model.