Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode.

The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5'-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.

[1]  Xiaodong Wang,et al.  R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway , 2003, Science.

[2]  Qinghua Liu,et al.  Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. , 2005, Genes & development.

[3]  B. Bass,et al.  RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. , 2006, RNA.

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

[5]  J. Choe,et al.  Mutational analysis of the hepatitis C virus RNA helicase , 1997, Journal of virology.

[6]  A. Pyle.  Translocation and unwinding mechanisms of RNA and DNA helicases. , 2008, Annual review of biophysics.

[7]  Taishin Kin,et al.  Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells , 2008, Nature.

[8]  W. Filipowicz,et al.  Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP , 2002, The EMBO journal.

[9]  Scott Kennedy,et al.  Requirement for the ERI/DICER Complex in Endogenous RNA Interference and Sperm Development in Caenorhabditis elegans , 2009, Genetics.

[10]  Z. Weng,et al.  Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells , 2008, Science.

[11]  Qinghua Liu,et al.  Expression, purification, and analysis of recombinant Drosophila Dicer-1 and Dicer-2 enzymes. , 2008, Methods in molecular biology.

[12]  Trevis M. Alleyne,et al.  Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila , 2010, Nature Structural &Molecular Biology.

[13]  N. Perrimon,et al.  An endogenous small interfering RNA pathway in Drosophila , 2008, Nature.

[14]  D. Wigley,et al.  Structure and mechanism of helicases and nucleic acid translocases. , 2007, Annual review of biochemistry.

[15]  Ayelet T. Lamm,et al.  Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma. , 2010, Molecular cell.

[16]  E. Sontheimer,et al.  An inside job for siRNAs. , 2008, Molecular cell.

[17]  Angela N. Brooks,et al.  Structural Basis for Double-Stranded RNA Processing by Dicer , 2006, Science.

[18]  J. Michaelis,et al.  Mechanisms of nucleic acid translocases: lessons from structural biology and single-molecule biophysics. , 2007, Current opinion in structural biology.

[19]  I. MacRae,et al.  Ribonuclease revisited: structural insights into ribonuclease III family enzymes. , 2007, Current opinion in structural biology.

[20]  Jack F Kirsch,et al.  Autoinhibition of human dicer by its internal helicase domain. , 2008, Journal of molecular biology.

[21]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[22]  E. Lai,et al.  Endogenous RNA Interference Provides a Somatic Defense against Drosophila Transposons , 2008, Current Biology.

[23]  Xuemei Chen,et al.  Small RNAs and their roles in plant development. , 2009, Annual review of cell and developmental biology.

[24]  E. Sontheimer,et al.  Origins and Mechanisms of miRNAs and siRNAs , 2009, Cell.

[25]  Gary Ruvkun,et al.  A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans , 2004, Nature.

[26]  Christopher M. Player,et al.  Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans , 2006, Cell.

[27]  D. Moazed,et al.  Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi. , 2007, Molecular cell.

[28]  T. Lohman,et al.  Non-hexameric DNA helicases and translocases: mechanisms and regulation , 2008, Nature Reviews Molecular Cell Biology.

[29]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[30]  G. Ruvkun,et al.  Functional Proteomics Reveals the Biochemical Niche of C. elegans DCR-1 in Multiple Small-RNA-Mediated Pathways , 2006, Cell.

[31]  N. Sonenberg,et al.  Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF‐4A. , 1992, The EMBO journal.

[32]  D. Bartel,et al.  The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs , 2008, Nature.

[33]  I. MacRae,et al.  Structural determinants of RNA recognition and cleavage by Dicer , 2007, Nature Structural &Molecular Biology.

[34]  Pedro J. Batista,et al.  Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway , 2010, Proceedings of the National Academy of Sciences.

[35]  A. Reynolds,et al.  The contributions of dsRNA structure to Dicer specificity and efficiency. , 2005, RNA.

[36]  Diana S Chu,et al.  26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans , 2009, Proceedings of the National Academy of Sciences.

[37]  E. Sontheimer,et al.  Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways , 2004, Cell.

[38]  Noah C. Welker,et al.  Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs. , 2010, RNA.

[39]  Phillip D Zamore,et al.  RNAi: nature abhors a double-strand. , 2002, Current opinion in genetics & development.

[40]  Gurman Singh Pall,et al.  Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot , 2007, Nucleic acids research.

[41]  K. Collins,et al.  Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs , 2007, Nature Structural &Molecular Biology.