In vitro reconstitution of the human RISC-loading complex

Targeted gene silencing by RNAi requires the RNA-induced silencing complex (RISC), whose core component is the protein Argonaute (Ago) bound to a microRNA (miRNA) or an siRNA. In humans, Ago2 is loaded with miRNAs by the action of a specialized assembly called the RISC-loading complex (RLC), comprising the proteins Ago2, Dicer, and TRBP. Here we show that the human RLC assembles spontaneously in vitro from purified components. No cofactors or chaperones are required for the complex to form. The reconstituted RLC, containing one copy of each protein, has the dicing, slicing, guide-strand selection, and Ago2-loading activities observed for the endogenous RLC. Furthermore, once Ago2 is loaded with an miRNA, it tends to dissociate from the rest of the complex. These results lay the groundwork for future structural and functional dissection of RISC loading in humans.

[1]  J. Steitz,et al.  AU-Rich-Element-Mediated Upregulation of Translation by FXR1 and Argonaute 2 , 2007, Cell.

[2]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

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

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

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

[6]  C. Robinson,et al.  Tandem mass spectrometry reveals the quaternary organization of macromolecular assemblies. , 2006, Chemistry & biology.

[7]  C. Robinson,et al.  Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. , 2006, Journal of the American Chemical Society.

[8]  Gunter Meister,et al.  Argonaute proteins: mediators of RNA silencing. , 2007, Molecular cell.

[9]  J. Doudna,et al.  Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle. , 2001, Journal of molecular biology.

[10]  C. Robinson,et al.  A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. , 2002, Analytical chemistry.

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

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

[13]  N. Sonenberg,et al.  Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Tariq M Rana,et al.  RNA helicase A interacts with RISC in human cells and functions in RISC loading. , 2007, Molecular cell.

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

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

[17]  W. Filipowicz,et al.  Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer , 2004, EMBO reports.

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

[19]  Shinsei Minoshima,et al.  Identification of eight members of the Argonaute family in the human genome. , 2003, Genomics.

[20]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

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

[22]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

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

[24]  A. Caudy,et al.  A micrococcal nuclease homologue in RNAi effector complexes , 2003, Nature.

[25]  E. Sontheimer,et al.  A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila , 2004, Cell.