Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

BackgroundDICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.ResultsIn this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.ConclusionsThe two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

[1]  Sui Huang.  Faculty Opinions recommendation of An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA. , 2009 .

[2]  Robert Blelloch,et al.  Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. , 2008, Genes & development.

[3]  Y. Hayashizaki,et al.  Novel ELISA using intracellularly biotinylated antigen for detection of antibody following DNA immunization. , 2010, Japanese journal of infectious diseases.

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

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

[6]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

[7]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[8]  Jennifer A. Doudna,et al.  Structural insights into RNA Processing by the Human RISC-Loading Complex , 2009, Nature Structural &Molecular Biology.

[9]  Y. Hayashizaki,et al.  A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness. , 2010, Genome research.

[10]  Neena Mitter,et al.  Repertoire of Bovine miRNA and miRNA-Like Small Regulatory RNAs Expressed upon Viral Infection , 2009, PloS one.

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

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

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

[14]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[15]  J. Doudna,et al.  A three-dimensional view of the molecular machinery of RNA interference , 2009, Nature.

[16]  Kiyoshi Asai,et al.  CentroidFold: a web server for RNA secondary structure prediction , 2009, Nucleic Acids Res..

[17]  V. Kim,et al.  Biogenesis of small RNAs in animals , 2009, Nature Reviews Molecular Cell Biology.

[18]  Kiyoshi Asai,et al.  Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing , 2008, Proceedings of the National Academy of Sciences.

[19]  D. Bartel,et al.  Intronic microRNA precursors that bypass Drosha processing , 2007, Nature.

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

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

[22]  Satoshi Shibata,et al.  A High-Resolution Structure of the Pre-microRNA Nuclear Export Machinery , 2009, Science.

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

[24]  P. Khaitovich,et al.  Sequence features associated with microRNA strand selection in humans and flies , 2009, BMC Genomics.

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

[26]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[27]  R. Gregory,et al.  Many roads to maturity: microRNA biogenesis pathways and their regulation , 2009, Nature Cell Biology.

[28]  Oliver H. Tam,et al.  Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes , 2008, Nature.

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

[30]  Y. Sakaki,et al.  Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes , 2008, Nature.

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

[32]  C. Nusbaum,et al.  Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. , 2010, Genes & development.

[33]  Yoshihide Hayashizaki,et al.  Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin , 2011, RNA biology.

[34]  Jennifer A. Doudna,et al.  In vitro reconstitution of the human RISC-loading complex , 2008, Proceedings of the National Academy of Sciences.

[35]  Michael T. McManus,et al.  Expanded RNA-binding activities of mammalian Argonaute 2 , 2009, Nucleic acids research.

[36]  E. Lai,et al.  The Mirtron Pathway Generates microRNA-Class Regulatory RNAs in Drosophila , 2007, Cell.

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

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

[39]  Eugene Berezikov,et al.  Mammalian mirtron genes. , 2007, Molecular cell.

[40]  A. Covarrubias,et al.  First step in pre-miRNAs processing by human Dicer , 2009, Acta Pharmacologica Sinica.

[41]  W. Filipowicz,et al.  Dicer Is Associated with Ribosomal DNA Chromatin in Mammalian Cells , 2010, PloS one.

[42]  Phillip D. Zamore,et al.  Sorting of Drosophila Small Silencing RNAs , 2007, Cell.

[43]  Piotr Kozlowski,et al.  Structural basis of microRNA length variety , 2010, Nucleic Acids Res..

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

[45]  Erik J. Sontheimer,et al.  Assembly and function of RNA silencing complexes , 2005, Nature Reviews Molecular Cell Biology.

[46]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[47]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[48]  Z. Paroo,et al.  ATP-dependent human RISC assembly pathways , 2010, Nature Structural &Molecular Biology.

[49]  M. Amarzguioui,et al.  A role for human Dicer in pre-RISC loading of siRNAs , 2010, Nucleic acids research.