A C-terminal Silencing Domain in GW182 is Essential for MiRNA Function

Overview

Journal RNA

Specialty Molecular Biology

Date 2009 Apr 23

PMID 19383769

Citations 67

Authors

Ana Eulalio

Sigrun Helms

Christoph Fritzsch

Maria Fauser

Elisa Izaurralde

Affiliations

Soon will be listed here.

Abstract

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182-AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine-tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.

Citing Articles

A local translation program regulates centriole amplification in the airway epithelium.

Liu H, Li H, Jiang Z, Jin S, Song R, Yang Y Sci Rep. 2023; 13(1):7090.

PMID: 37127654 PMC: 10151349. DOI: 10.1038/s41598-023-34365-8.

GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells.

Ghosh S, Mukherjee K, Chakrabarty Y, Chatterjee S, Ghoshal B, Bhattacharyya S Mol Cell Biol. 2021; 41(5).

PMID: 33685914 PMC: 8088265. DOI: 10.1128/MCB.00483-20.

microRNA strand selection: Unwinding the rules.

Medley J, Panzade G, Zinovyeva A Wiley Interdiscip Rev RNA. 2020; 12(3):e1627.

PMID: 32954644 PMC: 8047885. DOI: 10.1002/wrna.1627.

Defining an evolutionarily conserved role of GW182 in circular RNA degradation.

Jia R, Xiao M, Li Z, Shan G, Huang C Cell Discov. 2019; 5:45.

PMID: 31636958 PMC: 6796862. DOI: 10.1038/s41421-019-0113-y.

KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182.

Sharma N, Majerciak V, Kruhlak M, Yu L, Kang J, Yang A Nucleic Acids Res. 2019; 47(17):9368-9385.

PMID: 31400113 PMC: 6755100. DOI: 10.1093/nar/gkz683.

References

Behm-Ansmant I, Rehwinkel J, Izaurralde E . MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb Symp Quant Biol. 2007; 71:523-30. DOI: 10.1101/sqb.2006.71.013. View

Chu C, Rana T . Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 2006; 4(7):e210. PMC: 1475773. DOI: 10.1371/journal.pbio.0040210. View

Liu J, Valencia-Sanchez M, Hannon G, Parker R . MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol. 2005; 7(7):719-23. PMC: 1855297. DOI: 10.1038/ncb1274. View

Lazzaretti D, Tournier I, Izaurralde E . The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins. RNA. 2009; 15(6):1059-66. PMC: 2685519. DOI: 10.1261/rna.1606309. View

Bhattacharyya S, Habermacher R, Martine U, Closs E, Filipowicz W . Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell. 2006; 125(6):1111-24. DOI: 10.1016/j.cell.2006.04.031. View

Schneider M, Najand N, Chaker S, Pare J, Haskins J, Hughes S . Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development. J Cell Biol. 2006; 174(3):349-58. PMC: 2064231. DOI: 10.1083/jcb.200512103. View

Till S, Lejeune E, Thermann R, Bortfeld M, Hothorn M, Enderle D . A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol. 2007; 14(10):897-903. DOI: 10.1038/nsmb1302. View

Ding L, Spencer A, Morita K, Han M . The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol Cell. 2005; 19(4):437-47. DOI: 10.1016/j.molcel.2005.07.013. View

Giraldez A, Mishima Y, Rihel J, Grocock R, van Dongen S, Inoue K . Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science. 2006; 312(5770):75-9. DOI: 10.1126/science.1122689. View

10.

Eulalio A, Tritschler F, Buttner R, Weichenrieder O, Izaurralde E, Truffault V . The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing. Nucleic Acids Res. 2009; 37(9):2974-83. PMC: 2685099. DOI: 10.1093/nar/gkp173. View

11.

Wu L, Fan J, Belasco J . MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A. 2006; 103(11):4034-9. PMC: 1449641. DOI: 10.1073/pnas.0510928103. View

12.

Eulalio A, Behm-Ansmant I, Izaurralde E . P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Biol. 2006; 8(1):9-22. DOI: 10.1038/nrm2080. View

13.

Eulalio A, Behm-Ansmant I, Schweizer D, Izaurralde E . P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol. 2007; 27(11):3970-81. PMC: 1900022. DOI: 10.1128/MCB.00128-07. View

14.

Eulalio A, Huntzinger E, Izaurralde E . GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat Struct Mol Biol. 2008; 15(4):346-53. DOI: 10.1038/nsmb.1405. View

15.

Eystathioy T, Chan E, Tenenbaum S, Keene J, Griffith K, Fritzler M . A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol Biol Cell. 2002; 13(4):1338-51. PMC: 102273. DOI: 10.1091/mbc.01-11-0544. View

16.

Ding L, Han M . GW182 family proteins are crucial for microRNA-mediated gene silencing. Trends Cell Biol. 2007; 17(8):411-6. DOI: 10.1016/j.tcb.2007.06.003. View

17.

Rehwinkel J, Behm-Ansmant I, Gatfield D, Izaurralde E . A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA. 2005; 11(11):1640-7. PMC: 1370850. DOI: 10.1261/rna.2191905. View

18.

Liu J, Rivas F, Wohlschlegel J, Yates 3rd J, Parker R, Hannon G . A role for the P-body component GW182 in microRNA function. Nat Cell Biol. 2005; 7(12):1261-6. PMC: 1804202. DOI: 10.1038/ncb1333. View

19.

Landthaler M, Gaidatzis D, Rothballer A, Chen P, Soll S, Dinic L . Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. 2008; 14(12):2580-96. PMC: 2590962. DOI: 10.1261/rna.1351608. View

20.

Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel J . Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol. 2005; 7(12):1267-74. DOI: 10.1038/ncb1334. View