InteractiveFly: GeneBrief

Ge-1: Biological Overview | References

BIOLOGICAL OVERVIEW

microRNAs (miRNAs) silence gene expression by suppressing protein production and/or by promoting mRNA decay. To elucidate how silencing is accomplished, an RNA interference library was screened for suppressors of miRNA-mediated regulation in Drosophila cells. In addition to proteins known to be required for miRNA biogenesis and function (i.e., Drosha, Pasha, Dicer-1, AGO1, and GW182), the screen identified the decapping activator Ge-1 as being required for silencing by miRNAs. Depleting Ge-1 alone and/or in combination with other decapping activators (e.g., DCP1, EDC3, HPat, or Me31B) suppresses silencing of several miRNA targets, indicating that miRNAs elicit mRNA decapping. The protein interaction profile of Ge-1 suggests that Ge-1 may act as a molecular scaffold bringing together DCP2 (a decapping enzyme that must bind RNA in order to recognize the cap for hydrolysis) and decapping activators, and possibly nucleating the assembly of P-bodies. A comparison of gene expression profiles in cells depleted of AGO1 or of individual decapping activators shows that ~15% of AGO1-targets are also regulated by Ge-1, DCP1, and HPat, whereas 5% are dependent on EDC3 and LSm1-7. These percentages are underestimated because decapping activators are partially redundant. Furthermore, in the absence of active translation, some miRNA targets are stabilized, whereas others continue to be degraded in a miRNA-dependent manner. These findings suggest that miRNAs mediate post-transcriptional gene silencing by more than one mechanism (Eulalio, 2007c).

Post-transcriptional gene regulation plays a central role in biological processes as diverse as development, differentiation, stress response, and growth control. In particular, regulations of mRNA half-lives and of translation are key mechanisms by which the expression of many genes can be rapidly changed in response to extracellular signals. Post-transcriptional regulation is often mediated by specific RNA-binding proteins that recognize control elements in the mRNA 3' untranslated regions (UTRs) and promote (or antagonize) mRNA degradation or translational repression. mRNA decay or translational repression can also be triggered by short interfering RNAs (siRNAs) or microRNAs (miRNAs), two classes of noncoding RNAs that associate with proteins of the Argonaute family, to regulate the expression of fully or partially complementary target mRNAs (Eulalio, 2007c).

Degradation of bulk mRNA in eukaryotes is normally initiated by the gradual shortening of the poly(A) tail (deadenylation). In Saccharomyces cerevisiae and Drosophila cells, the major deadenylase activity is associated with the CAF1:CCR4:NOT complex (Parker, 2004). Deadenylated mRNA can then be exonucleolytically digested from the 3' end by the exosome and cofactors (Parker, 2004). Alternatively, decay proceeds by the removal of the cap structure by the decapping enzyme DCP2 and subsequent 5'-to-3' exonucleolytic degradation by XRN1 (Parker, 2004; Eulalio, 2007c and references therein).

In S. cerevisiae, several proteins stimulate decapping by DCP2 including DCP1, EDC3 (enhancer of decapping 3), the LSm1-7 complex, Pat1, and the RNA helicase Dhh1. These proteins are generically termed decapping activators, although they may activate decapping by different mechanisms (Parker, 2004). In human cells, DCP2 is part of a multimeric protein complex that includes DCP1, EDC3, the RNA helicase RCK/p54 (the human ortholog of Dhh1), and Ge-1 (also known as human enhancer of decapping large subunit, Hedls), a protein that has no orthologs in S. cerevisiae (Fenger-Grøn, 2005; Yu, 2005; Eulalio, 2007c and references therein).

Recent studies in zebrafish embryos, Drosophila, and human cells have shown that miRNAs accelerate the deadenylation and decapping of their targets by recruiting components of the general mRNA degradation machinery (Bagga, 2005; Behm-Ansmant, 2006a; Behm-Ansmant, 2006b; Giraldez, 2006; Wu, 2006). miRNA-mediated mRNA decay requires the Argonaute proteins, the P-body component GW182, the CAF1:CCR4:NOT deadenylase complex, the decapping enzyme DCP2, and the decapping activator DCP1. Moreover, in human cells, the decapping activator RCK/p54 has been implicated in silencing by miRNAs (Eulalio, 2007c and references therein).

miRNAs not only promote mRNA degradation, but they can also repress protein production, often without detectable changes in the abundance of target mRNAs (for review, see Pillai, 2006; Jackson, 2007; Nilsen, 2007). Several mechanisms for the miRNA-mediated down-regulation of protein expression have been proposed. These include (1) cotranslational protein degradation, (2) inhibition of translation elongation, (3) premature termination of translation (ribosome drop-off), or (4) inhibition of translation initiation (for review, see Pillai, 2006; Jackson, 2007; Nilsen, 2007; Eulalio, 2007c and references therein).

Two recent reports demonstrated that miRNAs inhibit translation; surprisingly, each proposed a different underlying mechanism (Chendrimada, 2007; Kiriakidou, 2007). Kiriakidou showed that the human Argonaute-2 (AGO2) polypeptide sequence exhibits sequence similarities with the cytoplasmic cap-binding protein eIF4E (eukaryotic initiation factor 4E) and binds the mRNA cap structure. This supports the argument that miRNAs (in complex with human AGO2) can inhibit translation at the initiation step by competing with eIF4E for the cap structure. Chendrimada identified the eukaryotic initiation factor 6 (eIF6) as a binding partner of AGO2 in human cells. eIF6 interacts with the large ribosomal subunit and prevents its premature association with the small subunit. If AGO2 recruits eIF6 to miRNA targets, then it may repress translation at an early step by blocking association of the large ribosomal subunit (Chendrimada, 2007). Even though eIF6 is a translation factor, its depletion suppressed the silencing of targets regulated mainly at the mRNA level. The most straightforward interpretation of these results is that miRNAs silence genes primarily by inhibiting protein synthesis, and mRNA degradation is only a consequence of this primary event (Eulalio, 2007c and references therein).

To shed light on the mechanisms that allow miRNAs to repress expression of their targets, an RNA interference (RNAi) library was screened for suppressors of silencing mediated by miR-12. This screen identified genes already known to be required for miRNA biogenesis and function (Drosha, Pasha, Dicer-1, AGO1, and GW182), as well as the decapping activator Ge-1. Depleting Ge-1 alone and/or in combination with other decapping activators relieves silencing of targets by several miRNAs, indicating that miRNAs promote decapping and the subsequent mRNA degradation. Some miRNA targets are stabilized in the absence of active translation, whereas others are nevertheless degraded. These findings suggest the existence of more than one mechanism of miRNA function and may explain conflicting reports regarding the mechanisms of silencing by miRNAs (Eulalio, 2007c).

A screen was performed for suppressors of miRNA-mediated gene silencing using a reporter expressing the firefly luciferase (F-Luc) coding region with a 3' UTR from the D. melanogaster CG10011 transcript, an mRNA silenced by miR-12 (Rehwinkel, 2005; Rehwinkel, 2006; Behm-Ansmant, 2006a; Behm-Ansmant, 2006b). In the presence of miR-12, expression of both F-Luc protein and mRNA is reduced to similar extents, indicating that miR-12 directs this reporter for degradation. The screen was performed in duplicate with a double-stranded RNA (dsRNA) library that targets ~90% of all annotated genes in the Drosophila genome. This study identified 47 dsRNAs that relieved the silencing by miR-12, including six previously identified components of the D. melanogaster miRNA pathway; i.e., Drosha, Pasha, Dicer-1, AGO1, and GW182. Exportin-5, Loquacious, and eIF6, also implicated in miRNA biogenesis and function (Eulalio, 2007c).

Several new candidates were identified. To determine the specificity of the new candidates, the dsRNAs were resynthesized and tested using the same reporter either in the absence or presence of miR-12. When nonspecific effects of the dsRNAs on F-Luc-CG10011 expression (i.e., those observed in the absence of miR-12) were taken into account, only the dsRNA targeting Drosophila Ge-1 (CG6181) interfered specifically with silencing of the reporter (Eulalio, 2007c).

The Ge-1 protein family is characterized by four canonical N-terminal WD40 repeats (Deyholos, 2003 Fenger-Grøn, 2005; Yu, 2005; Xu, 2006). WD40 repeats are protein: protein interaction modules present in proteins with unrelated functions. These repeats adopt a six- to seven-blade propeller fold and usually require six to seven repeats for structural stability. Sequence alignments and secondary structure predictions of the region encompassing amino acids 136-558 of D. melanogaster Ge-1 revealed three additional WD40 repeats, probably adapted to a specialized function in this protein family; thus standard computer programs might not detect statistically significant sequence similarities to known WD40s. The Ge-1 protein family is also characterized by a conserved C-terminal domain containing a highly conserved motif. The N-terminal β-propeller and the C-terminal domains are separated by a low-complexity region rich in serines (S-rich linker) that probably provides a flexible linker between these two protein domains (Eulalio, 2007c).

The Ge-1 proteins in Homo sapiens, D. melanogaster, and Arabidopsis thaliana localize to P-bodies and are required for P-body integrity (Fenger-Grøn, 2005; Yu, 2005; Xu, 2006; Eulalio, 2007a, Eulalio, 2007b). Analysis of the subcellular localization of HA-tagged Ge-1 protein domains (the N-terminal β-propeller domain, the S-rich linker, and the C-terminal domain) indicated that the C-terminal conserved domain (amino acids 944-1354) localizes to cytoplasmic foci, as with the full-length protein. These foci corresponded to endogenous P-bodies, because they were labeled with antibodies recognizing the protein Trailer hitch (Tral), an endogenous P-body marker in D. melanogaster (Eulalio, 2007a: Eulalio, 2007b). The N-terminal domain encompassing the WD40 repeats spread diffusely throughout the cytoplasm, whereas the S-rich linker domain accumulated within the nucleus. Overexpressing these protein fragments did not affect the integrity of endogenous P-bodies, as evidenced by the presence of foci stained with anti-Tral antibodies. Thus, Ge-1 is a conserved component of P-bodies (Eulalio, 2007c).

The results presented in this study indicate that P-body components are required for silencing of endogenous miRNA targets. These include Ge-1, Me31B, HPat, EDC3, and the LSm1-7 complex, all shown to enhance decapping (Parker, 2004; Fenger-Grøn, 2005; Xu, 2006). This study identified novel components of the miRNA pathway by screening a dsRNA library targeting nearly all annotated Drosophila genes for suppressors of silencing. In addition to known components of the miRNA pathway (i.e., Drosha, Pasha, Dicer-1, and AGO1), the screen identified two P-body components: GW182 and Ge-1. A role for GW182 in the miRNA pathway has been reported before. AGO1- and GW182-depleted cells exhibit strikingly similar expression profiles, indicating that GW182 functions in the miRNA pathway and is unlikely to have additional roles in general mRNA turnover. Indeed, GW182 regulates nearly all the AGO1-targets that are detectable by microarray (i.e., that change levels in AGO1-depleted cells) (Behm-Ansmant, 2006a). In contrast, comparing the gene expression profiles of AGO1- and Ge-1-depleted cells revealed that Ge-1 is required for silencing ~15% of AGO1-targets (76 of 498 mRNAs) in Drosophile (Eulalio, 2007c).

Ge-1 belongs to a protein family characterized by N-terminal WD40-repeats, a central S-rich linker, and a conserved C-terminal domain. Multiple sequence alignment of all proteins possessing these domains revealed two paralogs in Oryza sativa and A. thaliana (Varicose and Varicose-related, VCS and VCR, respectively), and a single ortholog in animals and fungi, with the exception of S. cerevisiae (Deyholos, 2003; Fenger-Grøn, 2005; Yu, 2005; Xu, 2006; Eulalio, 2007c and references therein).

The N-terminal WD40-repeat region of VCS is required for its interaction with DCP1, whereas the C-terminal domain is required for its oligomerization and interaction with DCP2 (Xu, 2006). These interactions stimulate the catalytic activity of DCP2 (Fenger-Grøn, 2005; Xu, 2006). Apart from the interaction with DCP2, the C-terminal domain of Ge-1 is necessary and sufficient to localize the protein to P-bodies, which is in turn required for P-body integrity (Yu, 2005). Since in human cells, Ge-1 interacts with DCP2, DCP1, EDC3, and RCK/p54 (Fenger-Grøn, 2005), this suggests that Ge-1 may act as a molecular scaffold bringing together DCP2 and decapping activators, and possibly nucleating the assembly of P-bodies. Understanding how various Ge-1 domains interact with mRNA decay enzymes and influence P-body integrity awaits the further biochemical characterization of these domains (Eulalio, 2007c).

The presence of Ge-1 in a multiprotein complex consisting of the decapping enzyme DCP2 and additional decapping activators including DCP1, EDC3, and RCK/p54 in human cells (Fenger-Grøn, 2005) prompted an investigation of the role of additional enhancers of decapping in the miRNA pathway. Comparing gene expression profiles in cells depleted of AGO1, DCP1, Ge-1, HPat, EDC3, or LSm1 showed that 15% of the transcripts that are up-regulated in cells depleted of AGO1 are also up-regulated in cells depleted of DCP1, Ge-1, or HPat; whereas in cells depleted of EDC3 or LSm1, only 5% show concordant changes. The fraction of AGO1 targets that decapping activators also regulated is underestimated when comparing expression profiles of cells depleted of the proteins individually, because these proteins have partially redundant functions in silencing. This is evidenced by the observation that the depletion of decapping activators, with the exception of Ge-1, suppresses silencing (above the twofold threshold that was defined as significant) only when they are codepleted with Ge-1 or additional activators. This observation also provides an explanation for why these proteins were not identified in this or previous screens (Eulalio, 2007c).

An important finding from these experiments is that even in cases in which the codepletion of decapping activators restored reporter mRNA levels, luciferase expression was not always restored, because the accumulated transcripts were deadenylated. Consequently, screens based on protein expression (luciferase or GFP) are very likely to overlook factors required for silencing (Eulalio, 2007c).

Transcripts up-regulated by both AGO1 and decapping activators are enriched in predicted and validated targets of a subset of miRNAs. This enrichment is relevant because transcripts exclusively regulated by decapping activators but not by AGO1 are not significantly enriched in miRNA-binding sites. A feature that distinguishes transcripts regulated by both AGO1 and decapping activators from transcripts exclusively regulated by AGO1 is that the former have longer 3' UTRs on average (721 nucleotides [nt] vs. 629 nt). Nevertheless, the specific features of miRNA targets that lead to the dependence of decapping activators for their silencing are unknown (Eulalio, 2007c).

The screen was performed with a reporter that is mainly regulated at the mRNA level. Analyzing of miRNA activity in cells treated with cycloheximide showed that degradation of this reporter by miR-12 requires ongoing translation. Therefore, one might expect to identify translation factors in the screen. However, there are many reasons why these factors may escape detection, including inefficient depletions or a general inhibition of translation affecting firefly and/or Renilla luciferase expression in such a way that the suppression of silencing is masked by nonspecific effects (Eulalio, 2007c).

In particular, eIF6 did not score positively in the screen. Whether two consecutive eIF6 knockdowns (instead of a single depletion as in the screen) and prolonged depletion times (9 d instead of 7 d) restored F-Luc-CG10011 expression was tested. Under these conditions (i.e., 90% of the endogenous protein was depleted), a partial suppression of silencing (an approximate twofold to 2.5-fold increase in firefly luciferase expression) was observed for F-Luc-CG10011 and several other reporters. Nevertheless, further studies are needed to elucidate the role of eIF6 in the miRNA pathway in D. melanogaster (Eulalio, 2007c).

The inhibition of translation by miRNAs is not always followed by mRNA decay. Conversely, this study shows that some targets are degraded in the absence of active translation in Drosophila cells. Similarly, in human cells and zebrafish embryos, it has been reported that miRNA targets that cannot be translated (e.g., because of the presence of a strong stem-loop in the 5' UTR) are nevertheless subject to deadenylation and subsequent decay (Mishima, 2006; Wu, 2006; Eulalio, 2007c and refernces therein).

These results indicate that miRNAs use at least three mechanisms to down-regulate their targets: (1) inhibition of protein production without a significant change in mRNA level (which can be achieved in different ways), (2) translation-dependent mRNA decay, or (3) translation-independent mRNA decay. Because at least two modes of regulation have been observed for targets of the same miRNA (i.e., miR-9b and miR-1), it is unlikely that these different modes are specified by the miRNA itself. Moreover, because the extent of miRNA regulation occurs over a wide range of magnitudes and differs for different miRNA:target pairs, a model is favored whereby the specific features and/or composition of the mRNP itself influence the outcome of miRNA regulation. This may explain the conflicting reports regarding the mechanisms of silencing by miRNAs. A major challenge for future studies will be to elucidate how mRNP features and/or composition influence miRNA regulation (Eulalio, 2007c).

Search PubMed for articles about Drosophila Ge-1

Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R. and Pasquinelli, A. E. (2005). Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122: 553-563. PubMed ID: 16122423

Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P. and Izaurralde, E. (2006a). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20: 1885-1898. PubMed ID: 16815998

Behm-Ansmant, I., Rehwinkel, J. and Izaurralde, E. (2006b). MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb. Symp. Quant. Biol. 71: 523-530. PubMed ID: 17381335

Chendrimada, T. P., Finn, K.J., Ji, X., Baillat, D., Gregory, R. I., Liebhaber, S. A., Pasquinelli, A. E. and Shiekhattar, R. (2007). MicroRNA silencing through RISC recruitment of eIF6. Nature 447: 823-828. PubMed ID: 17507929

Deyholos, M. K., Cavaness, G. F., Hall, B., King, E., Punwani, J., Van Norman, J. and Sieburth, L. E. 2003. VARICOSE, a WD-domain protein, is required for leaf blade development. Development 130: 6577-6588. PubMed ID: 14660546

Eulalio, A., Behm-Ansmant, I. and Izaurralde, E. (2007a). P bodies: At the crossroads of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol. 8: 9-22. PubMed ID: 17183357

Eulalio, A., Behm-Ansmant, I., Schweizer, D. and Izaurralde, E. (2007b). P-body formation is a consequence, not the cause of RNA-mediated gene silencing. Mol. Cell. Biol. 27: 3970-3981. PubMed ID: 17403906

Eulalio, A., et al., (2007)c. Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing. Genes Dev. 21(20): 2558-70. PubMed ID: 17901217

Fenger-Grøn, M., Fillman, C., Norrild, B. and Lykke-Andersen, J. (2005). Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol. Cell 20: 905-915. PubMed ID: 16364915

Giraldez, A.J., Mishima, Y., Rihel, J., Grocock, R.J., Van Dongen, S., Inoue, K., Enright, A.J. and Schier, A.F. (2006). Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312: 75-79. PubMed ID: 16484454

Jackson, R.J. and Standart, N. (2007). How do microRNAs regulate gene expression? Sci. STKE 2007(367):re1. PubMed ID: 17200520

Kiriakidou, M., Tan, G.S., Lamprinaki, S., De Planell-Saguer, M., Nelson, P. T. and Mourelatos, Z. (2007). An mRNA m(7)G cap binding-like motif within human Ago2 represses translation. Cell 129: 1141-1151. PubMed ID: 17524464

Mishima, Y., Giraldez, A. J., Takeda, Y., Fujiwara, T., Sakamoto, H., Schier, A. F. and Inoue, K. (2006). Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr. Biol. 16: 2135-2142. PubMed ID: 17084698

Nilsen, T.W. (2007). Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet. 23: 243-249. PubMed ID: 17368621

Parker, R. and Song, H. (2004). The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol. 11: 121-127. PubMed ID: 14749774

Pillai, R. S., Bhattacharyya, S. N. and Filipowicz, W. (2006). Repression of protein synthesis by miRNAs: How many mechanisms? Trends Cell Biol. 17: 118-126. PubMed ID: 17197185

Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (2005). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11: 1640-1647. PubMed ID: 16177138

Rehwinkel, J., Natalin, P., Stark, A., Brennecke, J., Cohen, S. M. and Izaurralde, E. (2006). Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster. Mol. Cell. Biol. 26: 2965-2975. PubMed ID: 16581772

Wu, L., Fan, J. and Belasco, J. G. (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. 103: 4034-4039. PubMed ID: 16495412

Xu, J., Yang, J. Y., Niu, Q. W. and Chua, N. H. (2006). Arabidopsis DCP2, DCP1 and VARICOSE form a decapping complex required for postembryonic development. Plant Cell 18: 3386-3398. PubMed ID: 17158604

Yu, J.H., Yang, W.H., Gulick, T., Bloch, K.D. and Bloch, D.B. (2005). Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body. RNA 11: 1795-1802. PubMed ID: 16314453

date revised: 4 January 2008

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