5′ isomiR variation is of functional and evolutionary importance

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

[1]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[2]  Xavier Estivill,et al.  A highly expressed miR-101 isomiR is a functional silencing small RNA , 2013, BMC Genomics.

[3]  Sanghyuk Lee,et al.  miRGator v3.0: a microRNA portal for deep sequencing, expression profiling and mRNA targeting , 2012, Nucleic Acids Res..

[4]  C. Bracken,et al.  IsomiRs--the overlooked repertoire in the dynamic microRNAome. , 2012, Trends in genetics : TIG.

[5]  Zhiping Weng,et al.  Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals , 2012, Cell.

[6]  Jennifer L. Clancy,et al.  Complexity of Murine Cardiomyocyte miRNA Biogenesis, Sequence Variant Expression and Function , 2012, PloS one.

[7]  David L. A. Wood,et al.  MicroRNAs and their isomiRs function cooperatively to target common biological pathways , 2011, Genome Biology.

[8]  Eugene Berezikov,et al.  Evolution of microRNA diversity and regulation in animals , 2011, Nature Reviews Genetics.

[9]  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.

[10]  David Galas,et al.  Complexity of the microRNA repertoire revealed by next-generation sequencing. , 2010, RNA.

[11]  Ana Kozomara,et al.  miRBase: integrating microRNA annotation and deep-sequencing data , 2010, Nucleic Acids Res..

[12]  Selene L. Fernandez-Valverde,et al.  Dynamic isomiR regulation in Drosophila development. , 2010, RNA.

[13]  S. Griffiths-Jones,et al.  Functional Shifts in Insect microRNA Evolution , 2010, Genome biology and evolution.

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

[15]  Frank Speleman,et al.  miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis , 2010, Nature Cell Biology.

[16]  P. Zamore,et al.  Small silencing RNAs: an expanding universe , 2009, Nature Reviews Genetics.

[17]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[18]  C. Burge,et al.  Most mammalian mRNAs are conserved targets of microRNAs. , 2008, Genome research.

[19]  T. Tuschl,et al.  Structure of the guide-strand-containing argonaute silencing complex , 2008, Nature.

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

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

[22]  Ryan D. Morin,et al.  Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. , 2008, Genome research.

[23]  J. Steitz,et al.  Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation , 2007, Science.

[24]  Zhenyu Xuan,et al.  A biochemical approach to identifying microRNA targets , 2007, Proceedings of the National Academy of Sciences.

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

[26]  Manolis Kellis,et al.  Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. , 2007, Genome research.

[27]  H. Horvitz,et al.  Most Caenorhabditis elegans microRNAs Are Individually Not Essential for Development or Viability , 2007, PLoS genetics.

[28]  George Easow,et al.  Isolation of microRNA targets by miRNP immunopurification. , 2007, RNA.

[29]  D. Baulcombe,et al.  miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii , 2007, Nature.

[30]  G. Meister,et al.  Identification of Human microRNA Targets From Isolated Argonaute Protein Complexes , 2007, RNA biology.

[31]  N. Rajewsky,et al.  The evolution of gene regulation by transcription factors and microRNAs , 2007, Nature Reviews Genetics.

[32]  C. Burge,et al.  The Widespread Impact of Mammalian MicroRNAs on mRNA Repression and Evolution , 2005, Science.

[33]  Wei Cui,et al.  Differentiation of Human Embryonic Stem Cells to Neural Lineages in Adherent Culture by Blocking Bone Morphogenetic Protein Signaling , 2005, Stem cells.

[34]  V. Kim.  MicroRNA biogenesis: coordinated cropping and dicing , 2005, Nature Reviews Molecular Cell Biology.

[35]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[36]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[37]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[38]  Benjamin M. Wheeler,et al.  The deep evolution of metazoan microRNAs , 2009, Evolution & development.