miRNA‐dependent gene silencing involving Ago2‐mediated cleavage of a circular antisense RNA

MicroRNAs (miRNAs) are ∼22 nt non‐coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non‐coding antisense transcripts in human cells. Specifically, we show that miR‐671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration‐Related protein 1 (CDR1) locus in an Ago2‐slicer‐dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

[1]  K. Morris,et al.  Small Interfering RNA-Induced Transcriptional Gene Silencing in Human Cells , 2004, Science.

[2]  Joshua J. Forman,et al.  A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence , 2008, Proceedings of the National Academy of Sciences.

[3]  Masaki Kaibori,et al.  Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes , 2007, Hepatology.

[4]  D. Corey,et al.  Antisense transcripts are targets for activating small RNAs , 2008, Nature Structural &Molecular Biology.

[5]  A. G. de Herreros,et al.  A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial-mesenchymal transition. , 2008, Genes & development.

[6]  U. A. Ørom,et al.  MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. , 2008, Molecular cell.

[7]  S. Raguz,et al.  Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. , 2010, Molecular cell.

[8]  William R. Jeck,et al.  Expression of Linear and Novel Circular Forms of an INK4/ARF-Associated Non-Coding RNA Correlates with Atherosclerosis Risk , 2010, PLoS genetics.

[9]  K. Morris,et al.  Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells , 2009, Nucleic acids research.

[10]  John J Rossi,et al.  Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells , 2006, Nature Structural &Molecular Biology.

[11]  W. Filipowicz,et al.  Inhibition of Translational Initiation by Let-7 MicroRNA in Human Cells , 2005, Science.

[12]  K. Morris,et al.  Bidirectional Transcription Directs Both Transcriptional Gene Activation and Suppression in Human Cells , 2008, PLoS genetics.

[13]  T. Gingeras.  Implications of chimaeric non-co-linear transcripts , 2009, Nature.

[14]  M. Rosenblum,et al.  Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Morgan,et al.  Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of β-secretase , 2008, Nature Medicine.

[16]  S. Napoli,et al.  Promoter‐specific transcriptional interference and c‐myc gene silencing by siRNAs in human cells , 2009, The EMBO journal.

[17]  Hui Zhou,et al.  Deep Sequencing of Human Nuclear and Cytoplasmic Small RNAs Reveals an Unexpectedly Complex Subcellular Distribution of miRNAs and tRNA 3′ Trailers , 2010, PloS one.

[18]  C. Mayr,et al.  Widespread Shortening of 3′UTRs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells , 2009, Cell.

[19]  W. Filipowicz,et al.  Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. , 2004, Nucleic acids research.

[20]  Yvonne Tay,et al.  MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation , 2008, Nature.

[21]  J. Herman,et al.  Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation , 2005, Nature Genetics.

[22]  S. Batalov,et al.  Antisense Transcription in the Mammalian Transcriptome , 2005, Science.

[23]  T. Jensen,et al.  The exosome: a multipurpose RNA-decay machine. , 2008, Trends in biochemical sciences.

[24]  D. Pisano,et al.  Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs , 2008, Cell cycle.

[25]  J. Kjems,et al.  Improved silencing properties using small internally segmented interfering RNAs , 2007, Nucleic acids research.

[26]  M. Esteller,et al.  DNA methylomes, histone codes and miRNAs: tying it all together. , 2009, The international journal of biochemistry & cell biology.

[27]  P. Molloy,et al.  Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA-DNA heteroduplex substrates. , 2010, The Biochemical journal.

[28]  A. Feinberg,et al.  Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA , 2008, Nature.

[29]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[30]  K. Morris,et al.  Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells , 2007, Proceedings of the National Academy of Sciences.

[31]  A. Verdel,et al.  Common themes in siRNA-mediated epigenetic silencing pathways. , 2009, The International journal of developmental biology.

[32]  Kenneth A Howard,et al.  RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[33]  Yao-Tseng Chen,et al.  Cloning of a brain protein identified by autoantibodies from a patient with paraneoplastic cerebellar degeneration. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Jerry Pelletier,et al.  Short RNAs repress translation after initiation in mammalian cells. , 2006, Molecular cell.

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

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

[37]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[38]  P. Sætrom,et al.  MicroRNA-directed transcriptional gene silencing in mammalian cells , 2008, Proceedings of the National Academy of Sciences.

[39]  A. Pasquinelli,et al.  MicroRNA silencing through RISC recruitment of eIF6 , 2007, Nature.

[40]  A. Riggs,et al.  The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. , 2005, RNA.

[41]  David I. K. Martin,et al.  MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[42]  D. Corey,et al.  Involvement of AGO1 and AGO2 in mammalian transcriptional silencing , 2006, Nature Structural &Molecular Biology.

[43]  M. Rush,et al.  Selective trapping of circular double-stranded DNA molecules in solidifying agarose. , 1982, Plasmid.

[44]  Peter Goodfellow,et al.  Circular transcripts of the testis-determining gene Sry in adult mouse testis , 1993, Cell.

[45]  C. Rivetti,et al.  Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy , 2006, Nucleic acids research.

[46]  P. Pandolfi,et al.  A coding-independent function of gene and pseudogene mRNAs regulates tumour biology , 2010, Nature.

[47]  G. Wang,et al.  Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells* , 2005, Journal of Biological Chemistry.

[48]  F. Rosenbauer,et al.  PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element. , 2008, Genes & development.

[49]  E. Wentzel,et al.  A Hexanucleotide Element Directs MicroRNA Nuclear Import , 2007, Science.

[50]  Henning Urlaub,et al.  Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi , 2002, Cell.

[51]  John Zaunders,et al.  Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region , 2005, Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research.