Systematic identification of microRNA functions by combining target prediction and expression profiling

Target predictions and validations are major obstacles facing microRNA (miRNA) researchers. Animal miRNA target prediction is challenging because of limited miRNA sequence complementarity to the targets. In addition, only a small number of predicted targets have been experimentally validated and the miRNA mechanism is poorly understood. Here we present a novel algorithm for animal miRNA target prediction. The algorithm combines relevant parameters for miRNA target recognition and heuristically assigns different weights to these parameters according to their relative importance. A score calculation scheme is introduced to reflect the strength of each parameter. We also performed microarray time course experiments to identify downregulated genes due to miRNA overexpression. The computational target prediction is combined with the miRNA transfection experiment to systematically identify the gene targets of human miR-124. miR-124 overexpression led to a significant downregulation of many cell cycle related genes. This may be the result of direct suppression of a few cell growth inhibitors at the early stage of miRNA overexpression, and these targeted genes were continuously suppressed over a long period of time. Our high-throughput approach can be generalized to globally identify the targets and functions of other miRNAs.

[1]  The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis. , 1998, Blood.

[2]  E. Lai.  Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation , 2002, Nature Genetics.

[3]  R. Liem,et al.  Protein products of human Gas2-related genes on chromosomes 17 and 22 (hGAR17 and hGAR22) associate with both microfilaments and microtubules , 2003, Journal of Cell Science.

[4]  Anton J. Enright,et al.  MicroRNA targets in Drosophila , 2003, Genome Biology.

[5]  The GeneChip.  Design and Performance of the GeneChip ® Human Genome U 133 Plus 2 . 0 and Human Genome U 133 A 2 . 0 Arrays , 2003 .

[6]  C. Burge,et al.  The microRNAs of Caenorhabditis elegans. , 2003, Genes & development.

[7]  Julius Brennecke,et al.  Identification of Drosophila MicroRNA Targets , 2003, PLoS biology.

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

[9]  Ivo L. Hofacker,et al.  Vienna RNA secondary structure server , 2003, Nucleic Acids Res..

[10]  B. Seed,et al.  A PCR primer bank for quantitative gene expression analysis. , 2003, Nucleic acids research.

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

[12]  A. Hatzigeorgiou,et al.  A combined computational-experimental approach predicts human microRNA targets. , 2004, Genes & development.

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

[14]  Terry Gaasterland,et al.  Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets , 2004, Genome Biology.

[15]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature Reviews Genetics.

[16]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

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

[18]  Diana V. Dugas,et al.  MicroRNA regulation of gene expression in plants. , 2004, Current opinion in plant biology.

[19]  F. Slack,et al.  RAS Is Regulated by the let-7 MicroRNA Family , 2005, Cell.

[20]  Y. Li,et al.  Incorporating structure to predict microRNA targets. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Gerald M Rubin,et al.  Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. , 2005, Genes & development.

[22]  Kristin C. Gunsalus,et al.  microRNA Target Predictions across Seven Drosophila Species and Comparison to Mammalian Targets , 2005, PLoS Comput. Biol..

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

[24]  S. Shirazi-Beechey,et al.  The human colonic monocarboxylate transporter Isoform 1: its potential importance to colonic tissue homeostasis. , 2005, Gastroenterology.

[25]  James M. Pipas,et al.  SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells , 2005, Nature.

[26]  J. Castle,et al.  Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs , 2005, Nature.

[27]  R. Russell,et al.  Animal MicroRNAs Confer Robustness to Gene Expression and Have a Significant Impact on 3′UTR Evolution , 2005, Cell.

[28]  E. Miska,et al.  How microRNAs control cell division, differentiation and death. , 2005, Current opinion in genetics & development.

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

[30]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

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

[32]  A. Saïb,et al.  A Cellular MicroRNA Mediates Antiviral Defense in Human Cells , 2005, Science.

[33]  Kathryn A. O’Donnell,et al.  c-Myc-regulated microRNAs modulate E2F1 expression , 2005, Nature.

[34]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[35]  Shuang Huang,et al.  Involvement of MicroRNA in AU-Rich Element-Mediated mRNA Instability , 2005, Cell.

[36]  A. Pasquinelli,et al.  Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation , 2005, Cell.

[37]  Richard W. Carthew,et al.  Silence from within: Endogenous siRNAs and miRNAs , 2005, Cell.