The Chicken or the Egg: MicroRNA-Mediated Regulation of mRNA Translation or mRNA Stability
2009; Elsevier BV; Volume: 35; Issue: 6 Linguagem: Inglês
10.1016/j.molcel.2009.09.003
ISSN1097-4164
AutoresArina D. Omer, Maja M. Janas, Carl D. Novina,
Tópico(s)RNA modifications and cancer
ResumoIn this issue of Molecular Cell, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar demonstrate that in cell-free extracts from mouse Krebs-2 ascites, microRNA-mediated translational repression precedes target mRNA deadenylation, and identify GW182, PABP, and deadenylase subunits CAF1 and CCR4 as factors required for deadenylation. In this issue of Molecular Cell, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar demonstrate that in cell-free extracts from mouse Krebs-2 ascites, microRNA-mediated translational repression precedes target mRNA deadenylation, and identify GW182, PABP, and deadenylase subunits CAF1 and CCR4 as factors required for deadenylation. MicroRNAs (miRNAs) are abundant ∼22 nucleotide-long endogenous RNAs that have profound impact on eukaryotic biology by mechanisms that are incompletely understood. miRNAs guide protein complexes (miRNPs) to partially complementary target mRNAs and downregulate their expression by at least two mechanisms: repression of mRNA translation and acceleration of mRNA degradation. The earliest insights into miRNA function obtained in worms indicate that miRNA repression is exerted at the level of translation after initiation, without target mRNA destabilization (Olsen and Ambros, 1999Olsen P.H. Ambros V. Dev. Biol. 1999; 216: 671-680Crossref PubMed Scopus (950) Google Scholar, Seggerson et al., 2002Seggerson K. Tang L. Moss E.G. Dev. Biol. 2002; 243: 215-225Crossref PubMed Scopus (298) Google Scholar). However, more recent data indicate that miRNAs can also repress translation during initiation in flies and mammals, and promote mRNA decay through deadenylation in worms, flies, and mammals (reviewed by Filipowicz et al., 2008Filipowicz W. Bhattacharyya S.N. Sonenberg N. Nat. Rev. Genet. 2008; 9: 102-114Crossref PubMed Scopus (4152) Google Scholar). It is clear that target mRNAs can be repressed exclusively at the level of translation, exclusively at the level of mRNA stability, or by a combination of both mechanisms. But what determines the extent to which each process contributes to miRNA-mediated repression? Is mRNA deadenylation a cause or a consequence of translational inhibition? If it is a consequence, are the factors required to mediate deadenylation specifically recruited by the miRNP, or does the untranslated mRNA enter a default degradation pathway? If so, is the miRNP associated with translationally repressed mRNAs identical to the miRNP associated with deadenylated mRNAs? To begin to address these questions, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar use cell-free reactions in Krebs-2 mouse ascites (Mathonnet et al., 2007Mathonnet G. Fabian M.R. Svitkin Y.V. Parsyan A. Huck L. Murata T. Biffo S. Merrick W.C. Darzynkiewicz E. Pillai R.S. et al.Science. 2007; 317: 1764-1767Crossref PubMed Scopus (407) Google Scholar) and a reporter repressed by endogenous let-7 miRNA to demonstrate that miRNA-mediated translational repression and deadenylation are independent and sequential processes. In this report, translational repression occurs within the first hour, whereas target mRNA deadenylation begins in the second hour of the repression reaction. Translational repression in this extract requires a physiological cap structure and the cap-binding factor eIF4E, but deadenylation does not. Deadenylation also does not require active translation or an open reading frame, but it does require miRNA-binding sites and a poly(A) tail on the target mRNA. In these reactions, 55% of miRNA-directed repression occurs fast (in the first 15 min of the reaction) by blocking the access of the target mRNA to the translation machinery, followed by an approximately 22% increase in repression caused by slow destabilization of the target mRNA by deadenylation. Thus, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar establish the extent and temporal order of miRNA-dependent repression pathways. Next, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar identify factors that mediate the observed deadenylation of the target mRNA. The central effectors of miRNA-containing complexes, the Argonaute (Ago) proteins, do not directly mediate mRNA degradation except when the miRNA has perfect sequence complementarity to target mRNAs. Instead, Agos bound to miRNAs serve as a scaffold for the recruitment of various factors that mediate repression of mRNA targets. A key factor implicated in miRNA-mediated mRNA target destabilization is the P body protein GW182. Typically associated with P bodies, it is believed that GW182 may exchange with cytoplasmic proteins (not present in P bodies) as P bodies are remodeled during the cell cycle (Eulalio et al., 2007Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (749) Google Scholar). Consistent with observations in flies (Eulalio et al., 2009Eulalio A. Huntzinger E. Nishihara T. Rehwinkel J. Fauser M. Izaurralde E. RNA. 2009; 15: 21-32Crossref PubMed Scopus (333) Google Scholar), Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar demonstrate that the Ago2-GW182 interaction is important for miRNA-mediated deadenylation in vitro. Additionally, they characterize a new RNA-independent interaction between the poly(A)-binding protein, PABP, and the C terminus of GW182 that allows docking of the deadenylase complex. These data are inconsistent with studies performed in cells indicating that full-length GW182 coprecipitates with Agos but not PABP or deadenylase subunits. The reasons for these differences are not clear at present. There may be more than one mechanism by which deadenylases are recruited to target mRNAs. It is also possible that subsets of miRNAs use distinct mechanisms to destabilize the mRNA target as determined by the biological context. It will be important to elucidate the molecular details of these processes in future studies. CCR4/CAF1/NOT is a 1.2 MDa complex composed of approximately ten subunits that is one of three enzyme complexes responsible for mRNA deadenylases in eukaryotes. CCR4/CAF1/NOT acts sequentially with PAN2/PAN3 complexes to mediate 3′-5′ exonucleolytic degradation of poly(A) tails. Interestingly, Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar identify CAF1 and CCR4 deadenylase subunits by mass spectrometry in Ago1 and Ago2 immunoprecipitates from human HEK293 cells. These observations are consistent with data in flies indicating that more than half of Ago-silenced mRNA targets are coregulated by CAF1 deadenylase complexes (Eulalio et al., 2009Eulalio A. Huntzinger E. Nishihara T. Rehwinkel J. Fauser M. Izaurralde E. RNA. 2009; 15: 21-32Crossref PubMed Scopus (333) Google Scholar) and suggest that deadenylases may be recruited to target mRNAs by docking onto PABP independently of Agos or may be recruited to target mRNAs in a complex containing Agos. It is not clear whether deadenylases directly interact with Agos or GW182. It will be important to investigate the molecular details of the recruitment of CAF1-containing deadenylation complexes to target mRNAs. It is clear that in mammals miRNA-mediated translational repression requires a poly(A) tail (Humphreys et al., 2005Humphreys D.T. Westman B.J. Martin D.I. Preiss T. Proc. Natl. Acad. Sci. USA. 2005; 102: 16961-16966Crossref PubMed Scopus (488) Google Scholar, Wang et al., 2006Wang B. Love T.M. Call M.E. Doench J.G. Novina C.D. Mol. Cell. 2006; 22: 553-560Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and, in turn, PABP. But PABP has intricate multipurpose functions. It stabilizes "closed loop" mRNA conformation to facilitate ribosome joining and translation initiation and also protects the mRNA against degradation (Kahvejian et al., 2005Kahvejian A. Svitkin Y.V. Sukarieh R. M'Boutchou M.N. Sonenberg N. Genes Dev. 2005; 19: 104-113Crossref PubMed Scopus (360) Google Scholar). Paradoxically, PABP is also required for P body-associated functions: general mRNA degradation via deadenylation (Bernstein et al., 1989Bernstein P. Peltz S.W. Ross J. Mol. Cell. Biol. 1989; 9: 659-670Crossref PubMed Scopus (293) Google Scholar) and, as demonstrated by Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar in this study, miRNA-dependent deadenylation, even though PABP is not believed to accumulate in P bodies. The data reported by Fabian et al., 2009Fabian M.R. Mathonnet G. Sundermeier T. Mathys H. Zipprich J.T. Svitkin Y.V. Rivas F. Jinek M. Wohlschlegel J. Doudna J.A. et al.Mol. Cell. 2009; 35 (this issue): 868-880Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar support a model in which initial miRNA-mediated translational repression is followed by deadenylation that reinforces the translational repression. Interestingly, a report published recently suggests just the opposite: miRNAs mediate deadenylation first, followed by translational repression (Beilharz et al., 2009Beilharz T.H. Humphreys D.T. Clancy J.L. Thermann R. Martin D.I. Hentze M.W. Preiss T. PLoS ONE. 2009; 4: e6783https://doi.org/10.1371/journal.pone.0006783Crossref PubMed Scopus (75) Google Scholar); this report also confirms previous observations that increasing poly(A) tail length increases miRNA-mediated translational repression (Wang et al., 2006Wang B. Love T.M. Call M.E. Doench J.G. Novina C.D. Mol. Cell. 2006; 22: 553-560Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this scenario, target mRNA deadenylation would decrease translational repression. One way to integrate these data with previous observations is depicted in Figure 1. In this model, mRNAs targeted by miRNAs undergo translational repression effected by interactions between Ago- and PABP-containing complexes that disrupt the closed loop mRNA conformation, leading to poly(A) tail destabilization. These repressed mRNAs are translocated to P bodies where they are destabilized by deadenylation and either degraded to completion or, through events that are poorly defined, temporarily stored before re-entering active translation in the cytoplasm. This cyclic model may explain why some studies indicate that translational repression precedes deadenylation, whereas others indicate opposite order. A solution to this chicken or egg paradox in the temporal order of repressive events driven by miRNAs will require a detailed characterization of the components, interactions, and subcellular structures associated with miRNA function. Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent DeadenylationFabian et al.Molecular CellAugust 27, 2009In BriefMicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3′UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. Full-Text PDF Open Archive
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