Affinity purification of microRNA-133a with the cardiac transcription factor, Hand2

Predictions of microRNA-mRNA interactions typically rely on bioinformatic algorithms, but these algorithms only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. We developed an affinity purification approach to empirically identify microRNAs associated with the 3′UTR of the mRNA encoding Hand2, a transcription factor essential for cardiac development. In addition to miR-1, a known regulator of Hand2 expression, we determined that the Hand2 3′UTR also associated with miR-133a, a microRNA cotranscribed with miR-1 in cardiac and muscle cells. Using a sequential binding assay, we showed that miR-1 and miR-133a could occupy the Hand2 3′UTR concurrently. miR-133a inhibited Hand2 expression in tissue culture models, and miR-133a double knockout mice had elevated levels of Hand2 mRNA and protein. We conclude that Hand2 is regulated by miR-133a in addition to miR-1. The affinity purification assay should be generally applicable for identifying other microRNA-mRNA interactions.

[1]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[2]  Oliver Hofmann,et al.  miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to "seedless" 3'UTR microRNA recognition elements. , 2009, Molecular cell.

[3]  F. Noubissi,et al.  CRD-BP protects the coding region of betaTrCP1 mRNA from miR-183-mediated degradation. , 2009, Molecular cell.

[4]  A. Mele,et al.  Ago HITS-CLIP decodes miRNA-mRNA interaction maps , 2009, Nature.

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

[6]  E. Olson,et al.  microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart. , 2008, Genes & development.

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

[8]  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.

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

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

[11]  Oliver Hobert,et al.  Molecular architecture of a miRNA-regulated 3' UTR. , 2008, RNA.

[12]  Michael D. Schneider,et al.  Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure , 2008, Proceedings of the National Academy of Sciences.

[13]  Reuven Agami,et al.  RNA-Binding Protein Dnd1 Inhibits MicroRNA Access to Target mRNA , 2007, Cell.

[14]  M. Latronico,et al.  Emerging role of microRNAs in cardiovascular biology. , 2007, Circulation research.

[15]  A. Lund,et al.  Isolation of microRNA targets using biotinylated synthetic microRNAs. , 2007, Methods.

[16]  E. Olson,et al.  MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. , 2007, The Journal of clinical investigation.

[17]  Chaoqian Xu,et al.  The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes , 2007, Journal of Cell Science.

[18]  L. Lim,et al.  MicroRNA targeting specificity in mammals: determinants beyond seed pairing. , 2007, Molecular cell.

[19]  Michael T. McManus,et al.  Dysregulation of Cardiogenesis, Cardiac Conduction, and Cell Cycle in Mice Lacking miRNA-1-2 , 2007, Cell.

[20]  Oliver Hobert,et al.  Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions , 2006, Nature Structural &Molecular Biology.

[21]  Harvey F Lodish,et al.  Myogenic factors that regulate expression of muscle-specific microRNAs. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Jian-Fu Chen,et al.  The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation , 2006, Nature Genetics.

[23]  Olivier Elemento,et al.  Revealing Posttranscriptional Regulatory Elements Through Network-Level Conservation , 2005, PLoS Comput. Biol..

[24]  Yong Zhao,et al.  Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis , 2005, Nature.

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

[26]  R. Russell,et al.  Principles of MicroRNA–Target Recognition , 2005, PLoS biology.

[27]  Steven P. Gygi,et al.  Comprehensive proteomic analysis of the human spliceosome , 2002, Nature.

[28]  David Baltimore,et al.  Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors , 2002, Science.

[29]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.

[30]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.