RNA helicase A interacts with RISC in human cells and functions in RISC loading.

RNA interference is a conserved pathway of sequence-specific gene silencing that depends on small guide RNAs and the action of proteins assembled in the RNA-induced silencing complex (RISC). Minimally, the action of RISC requires the endonucleolytic slicer activity of Argonaute2 (Ago2) directed to RNA targets whose sequences are complementary to RISC-incorporated small RNA. To identify RISC components in human cells, we developed an affinity-purification strategy to isolate siRNA-programmed RISC. Here we report the identification of RNA helicase A (RHA) as a human RISC-associated factor. We show that RHA interacts in human cells with siRNA, Ago2, TRBP, and Dicer and functions in the RNAi pathway. In RHA-depleted cells, RNAi was reduced as a consequence of decreased intracellular concentration of active RISC assembled with the guide-strand RNA and Ago2. Our results identify RHA as a RISC component and demonstrate that RHA functions in RISC as an siRNA-loading factor.

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

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

[3]  Cheryl Bolinger,et al.  RNA helicase A is necessary for translation of selected messenger RNAs , 2006, Nature Structural &Molecular Biology.

[4]  Sangdun Choi,et al.  Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy , 2005, Nature Biotechnology.

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

[6]  Ji-Joon Song,et al.  Purified Argonaute2 and an siRNA form recombinant human RISC , 2005, Nature Structural &Molecular Biology.

[7]  M. Mann,et al.  A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II , 2001, The Journal of cell biology.

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

[9]  P. Linder.  Dead-box proteins: a family affair—active and passive players in RNP-remodeling , 2006, Nucleic acids research.

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

[11]  V. Kim,et al.  The role of PACT in the RNA silencing pathway , 2006, The EMBO journal.

[12]  Wei Ge,et al.  Synthetic shRNAs as potent RNAi triggers , 2005, Nature Biotechnology.

[13]  R. Jackups,et al.  Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. , 2001, Molecular cell.

[14]  J. Lucchesi,et al.  Dosage compensation in flies and worms: the ups and downs of X-chromosome regulation. , 1998, Current opinion in genetics & development.

[15]  T. Rana,et al.  Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54 , 2006, PLoS biology.

[16]  D. Rose,et al.  A role for RNA helicase A in post-transcriptional regulation of HIV type 1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Peters,et al.  Modular Structure of PACT: Distinct Domains for Binding and Activating PKR , 2001, Molecular and Cellular Biology.

[18]  J. Hurwitz,et al.  Human RNA helicase A is homologous to the maleless protein of Drosophila. , 1993, The Journal of biological chemistry.

[19]  S. Baylin,et al.  Sequence-specific DNA Binding Activity of RNA Helicase A to thep16INK4a Promoter* , 2001, The Journal of Biological Chemistry.

[20]  Rekha C. Patel,et al.  The carboxy-terminal, M3 motifs of PACT and TRBP have opposite effects on PKR activity. , 2003, Virology.

[21]  U. Kutay,et al.  Nuclear Export of MicroRNA Precursors , 2004, Science.

[22]  A. Reynolds,et al.  Rational siRNA design for RNA interference , 2004, Nature Biotechnology.

[23]  K. Manova,et al.  RNA helicase A is essential for normal gastrulation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Phillip D Zamore,et al.  Perspective: machines for RNAi. , 2005, Genes & development.

[25]  David P. Bartel,et al.  Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes , 2005, Cell.

[26]  L. Maltais,et al.  The human DDX and DHX gene families of putative RNA helicases. , 2003, Genomics.

[27]  Jeffrey D. Parvin,et al.  RNA Helicase A Mediates Association of CBP with RNA Polymerase II , 1997, Cell.

[28]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[29]  P. Zamore,et al.  A Protein Sensor for siRNA Asymmetry , 2004, Science.

[30]  A. Pyle,et al.  A DEAD protein that activates intron self-splicing without unwinding RNA. , 2006, Molecular cell.

[31]  F. Grosse,et al.  Nuclear DNA Helicase II (RNA Helicase A) Interacts with Werner Syndrome Helicase and Stimulates Its Exonuclease Activity* , 2005, Journal of Biological Chemistry.

[32]  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.

[33]  T. Rana,et al.  Specific and potent RNAi in the nucleus of human cells , 2005, Nature Structural &Molecular Biology.

[34]  D. McDonald,et al.  The Carboxyl Terminus of RNA Helicase A Contains a Bidirectional Nuclear Transport Domain , 1999, Molecular and Cellular Biology.

[35]  Xiaodong Wang,et al.  Argonaute2 Cleaves the Anti-Guide Strand of siRNA during RISC Activation , 2005, Cell.

[36]  J. Parvin,et al.  BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A , 1998, Nature Genetics.

[37]  J. Hurwitz,et al.  A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. , 1992, The Journal of biological chemistry.

[38]  T. Du,et al.  RISC Assembly Defects in the Drosophila RNAi Mutant armitage , 2004, Cell.

[39]  T. Okamoto,et al.  RNA helicase A interacts with nuclear factor kappaB p65 and functions as a transcriptional coactivator. , 2004, European journal of biochemistry.

[40]  A. Safa,et al.  RNA Helicase A in the MEF1 Transcription Factor Complex Up-regulates the MDR1 Gene in Multidrug-resistant Cancer Cells* , 2004, Journal of Biological Chemistry.

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

[42]  Qiang Zhou,et al.  The Yin and Yang of P-TEFb Regulation: Implications for Human Immunodeficiency Virus Gene Expression and Global Control of Cell Growth and Differentiation , 2006, Microbiology and Molecular Biology Reviews.

[43]  T. Rana,et al.  Illuminating the silence: understanding the structure and function of small RNAs , 2007, Nature Reviews Molecular Cell Biology.

[44]  W. G. Kelly,et al.  RNA helicase A is important for germline transcriptional control, proliferation, and meiosis in C. elegans , 2005, Mechanisms of Development.

[45]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

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

[47]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[48]  M. Hagiwara,et al.  Dual Roles of RNA Helicase A in CREB-Dependent Transcription , 2001, Molecular and Cellular Biology.

[49]  Z. Mourelatos,et al.  A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. , 2005, Genes & development.

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

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

[52]  G. Sen,et al.  PACT, a protein activator of the interferon‐induced protein kinase, PKR , 1998, The EMBO journal.