Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

[1]  Kunio Inoue.  [MicroRNA function in animal development]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[2]  T. Rana,et al.  Human Retroviral Host Restriction Factors APOBEC3G and APOBEC3F Localize to mRNA Processing Bodies , 2006, PLoS pathogens.

[3]  Jerry Pelletier,et al.  Short RNAs repress translation after initiation in mammalian cells. , 2006, Molecular cell.

[4]  T. Tuschl,et al.  Identification of Novel Argonaute-Associated Proteins , 2005, Current Biology.

[5]  C. Gregory,et al.  Apoptosis: eating sensibly , 2005, Nature Cell Biology.

[6]  David I. K. Martin,et al.  MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[8]  E. Chan,et al.  Disruption of GW bodies impairs mammalian RNA interference , 2005, Nature Cell Biology.

[9]  J. Yates,et al.  A role for the P-body component GW182 in microRNA function , 2005, Nature Cell Biology.

[10]  Isabelle Behm-Ansmant,et al.  A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. , 2005, RNA.

[11]  Anne Gatignol,et al.  TRBP, a regulator of cellular PKR and HIV‐1 virus expression, interacts with Dicer and functions in RNA silencing , 2005, EMBO reports.

[12]  Roy Parker,et al.  General Translational Repression by Activators of mRNA Decapping , 2005, Cell.

[13]  N. Sonenberg,et al.  A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay , 2005, The Journal of cell biology.

[14]  Phillip D. Zamore,et al.  Ribo-gnome: The Big World of Small RNAs , 2005, Science.

[15]  W. Filipowicz,et al.  Inhibition of Translational Initiation by Let-7 MicroRNA in Human Cells , 2005, Science.

[16]  Min Han,et al.  The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. , 2005, Molecular cell.

[17]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[18]  W. Filipowicz,et al.  RNAi: The Nuts and Bolts of the RISC Machine , 2005, Cell.

[19]  J. Rossi.  RNAi and the P-body connection , 2005, Nature Cell Biology.

[20]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[21]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[22]  Gregory J. Hannon,et al.  MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies , 2005, Nature Cell Biology.

[23]  J. Carrington,et al.  Small RNAs and Arabidopsis. A Fast Forward Look , 2005, Plant Physiology.

[24]  T. Rana,et al.  Dissecting RNA-interference pathway with small molecules. , 2005, Chemistry & biology.

[25]  A. Denli,et al.  Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein , 2005, PLoS biology.

[26]  H. Blau,et al.  Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies , 2005, Nature Cell Biology.

[27]  R. Heintzmann,et al.  A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. , 2005, RNA.

[28]  Tariq M Rana,et al.  Target accessibility dictates the potency of human RISC , 2005, Nature Structural &Molecular Biology.

[29]  N. Sonenberg,et al.  Translational control in stress and apoptosis , 2005, Nature Reviews Molecular Cell Biology.

[30]  F. Slack,et al.  RAS Is Regulated by the let-7 MicroRNA Family , 2005, Cell.

[31]  N. Rajewsky,et al.  A pancreatic islet-specific microRNA regulates insulin secretion , 2004, Nature.

[32]  M. Hentze,et al.  Molecular mechanisms of translational control , 2004, Nature Reviews Molecular Cell Biology.

[33]  D. Baulcombe.  RNA silencing in plants , 2004, Nature.

[34]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[35]  G. Hannon,et al.  Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity , 2004, Science.

[36]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[37]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[38]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[39]  Roy Parker,et al.  Eukaryotic mRNA decapping. , 2004, Annual review of biochemistry.

[40]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[41]  B. Séraphin,et al.  Cytoplasmic foci are sites of mRNA decay in human cells , 2004, The Journal of cell biology.

[42]  N. Sonenberg,et al.  A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Thomas Tuschl,et al.  Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. , 2004, RNA.

[44]  Phillip D Zamore,et al.  Sequence-Specific Inhibition of Small RNA Function , 2004, PLoS biology.

[45]  N. Standart,et al.  The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer. , 2004, Nucleic acids research.

[46]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[47]  Eun-Young Choi,et al.  The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. , 2004, Genes & development.

[48]  T. Rana,et al.  siRNA function in RNAi: a chemical modification analysis. , 2003, RNA.

[49]  B. Cullen,et al.  MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Y. Akao,et al.  Overexpression of a DEAD box/RNA helicase protein, rck/p54, in human hepatocytes from patients with hepatitis C virus‐related chronic hepatitis and its implication in hepatocellular carcinogenesis , 2003, Journal of viral hepatitis.

[51]  A. Caudy,et al.  Fragile X-related protein and VIG associate with the RNA interference machinery. , 2002, Genes & development.

[52]  T. Rana,et al.  RNAi in human cells: basic structural and functional features of small interfering RNA. , 2002, Molecular cell.

[53]  W. Filipowicz.  Faculty Opinions recommendation of A microRNA in a multiple-turnover RNAi enzyme complex. , 2002 .

[54]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[55]  N. Standart,et al.  A conserved role of a DEAD box helicase in mRNA masking. , 2001, RNA.

[56]  R. Parker,et al.  The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. , 2001, RNA.

[57]  Y. Akao,et al.  Co-overexpression of DEAD box protein rck/p54 and c-myc protein in human colorectal adenomas and the relevance of their expression in cultured cell lines. , 2001, Carcinogenesis.

[58]  A. Nakamura,et al.  Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. , 2001, Development.

[59]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[60]  G. Patterson,et al.  Förster distances between green fluorescent protein pairs. , 2000, Analytical biochemistry.

[61]  Y. Akao,et al.  Overexpression of rck/p54, a DEAD box protein, in human colorectal tumours , 1999, British Journal of Cancer.

[62]  A. Kenworthy,et al.  Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer , 1998, The Journal of cell biology.

[63]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[64]  M. Ladomery,et al.  Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes. , 1997, Nucleic acids research.

[65]  T M Jovin,et al.  Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. , 1996, The EMBO journal.

[66]  Y. Akao,et al.  The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues. , 1995, Cancer research.

[67]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[68]  R. Clegg.  FRET tells us about proximities, distances, orientations and dynamic properties. , 2002, Journal of biotechnology.

[69]  R. Tsien,et al.  Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. , 2000, Methods in enzymology.

[70]  M. A. Rector,et al.  References and Notes Materials and Methods Som Text Fig. S1 Table S1 References a Microrna in a Multiple- Turnover Rnai Enzyme Complex , 2022 .