Canonical and alternate functions of the microRNA biogenesis machinery.

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.

[1]  Michael T. McManus,et al.  Dicer loss in striatal neurons produces behavioral and neuroanatomical phenotypes in the absence of neurodegeneration , 2008, Proceedings of the National Academy of Sciences.

[2]  David W. Taylor,et al.  A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity , 2010, Science.

[3]  D. Bartel,et al.  Microprocessor-independent , Dicer-dependent small RNAs Mouse ES cells express endogenous shRNAs , siRNAs , and other Material Supplemental , 2008 .

[4]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[5]  D. Haussler,et al.  Posttranscriptional Crossregulation between Drosha and DGCR8 , 2009, Cell.

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

[7]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

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

[9]  Michael T. McManus,et al.  The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[11]  Nectarios Koziris,et al.  DIANA-microT web server: elucidating microRNA functions through target prediction , 2009, Nucleic Acids Res..

[12]  D. Fenyö,et al.  Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer. , 2008, Journal of proteome research.

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

[14]  W. Filipowicz,et al.  Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? , 2008, Nature Reviews Genetics.

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

[16]  Mary Goldman,et al.  The UCSC Genome Browser database: update 2011 , 2010, Nucleic Acids Res..

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

[18]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[19]  Michael Q. Zhang,et al.  Critical roles for Dicer in the female germline. , 2007, Genes & development.

[20]  Sebastian Kadener,et al.  Genome-wide identification of targets of the drosha-pasha/DGCR8 complex. , 2009, RNA.

[21]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

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

[23]  A. Hata,et al.  Regulation of MicroRNA Biogenesis: A miRiad of mechanisms , 2009, Cell Communication and Signaling.

[24]  A. Rudensky,et al.  The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease , 2008, The Journal of Experimental Medicine.

[25]  C. Burge,et al.  Prediction of Mammalian MicroRNA Targets , 2003, Cell.

[26]  S. Yamanaka,et al.  Induction of pluripotent stem cells from fibroblast cultures , 2007, Nature Protocols.

[27]  D. Bartel,et al.  The impact of microRNAs on protein output , 2008, Nature.

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

[29]  F. Tang,et al.  Maternal microRNAs are essential for mouse zygotic development. , 2007, Genes & development.

[30]  V. Kim,et al.  In vitro and in vivo assays for the activity of Drosha complex. , 2007, Methods in enzymology.

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

[32]  N. Rajewsky,et al.  Widespread changes in protein synthesis induced by microRNAs , 2008, Nature.

[33]  H. Macdonald,et al.  Inactivation of Notch 1 in immature thymocytes does not perturb CD4 or CD8T cell development. , 2001, Nature immunology.

[34]  Thomas Tuschl,et al.  Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. , 2008, Methods.

[35]  G. Hannon,et al.  A dicer-independent miRNA biogenesis pathway that requires Ago catalysis , 2010, Nature.

[36]  H. Macdonald,et al.  Inactivation of Notch1 in immature thymocytes does not perturb CD4 or CD8 T cell development , 2001, Nature Immunology.

[37]  W. M. Weaver,et al.  A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. , 2001, Immunity.

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

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

[40]  R. Blelloch,et al.  Genomic Analysis Suggests that mRNA Destabilization by the Microprocessor Is Specialized for the Auto-Regulation of Dgcr8 , 2009, PloS one.

[41]  Gene W Yeo,et al.  RNA sequence analysis defines Dicer's role in mouse embryonic stem cells , 2007, Proceedings of the National Academy of Sciences.

[42]  V. Kim,et al.  The Drosha-DGCR8 complex in primary microRNA processing. , 2004, Genes & development.

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

[44]  Oliver H. Tam,et al.  Characterization of Dicer-deficient murine embryonic stem cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[45]  David Haussler,et al.  The UCSC Genome Browser database: update 2010 , 2009, Nucleic Acids Res..

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

[47]  C. Burge,et al.  A computational analysis of sequence features involved in recognition of short introns , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[48]  K. Gunsalus,et al.  Combinatorial microRNA target predictions , 2005, Nature Genetics.

[49]  S. Elledge,et al.  Dicer is essential for mouse development , 2003, Nature Genetics.