title: Comparative genomic analysis of upstream miRNA regulatory motifs in Caenorhabditis.
abstract: MicroRNAs (miRNAs) comprise a class of short noncoding RNA molecules that play diverse developmental and physiological roles by controlling mRNA abundance and protein output of the vast majority of transcripts. Despite the importance of miRNAs in regulating gene function, we still lack a complete understanding of how miRNAs themselves are transcriptionally regulated. To fill this gap, we predicted regulatory sequences by searching for abundant short motifs located upstream of miRNAs in eight species of Caenorhabditis nematodes. We identified three conserved motifs across the Caenorhabditis phylogeny that show clear signatures of purifying selection from comparative genomics, patterns of nucleotide changes in motifs of orthologous miRNAs, and correlation between motif incidence and miRNA expression. We then validated our predictions with transgenic green fluorescent protein reporters and site-directed mutagenesis for a subset of motifs located in an enhancer region upstream of let-7 We demonstrate that a CT-dinucleotide motif is sufficient for proper expression of GFP in the seam cells of adult C. elegans, and that two other motifs play incremental roles in combination with the CT-rich motif. Thus, functional tests of sequence motifs identified through analysis of molecular evolutionary signatures provide a powerful path for efficiently characterizing the transcriptional regulation of miRNA genes.
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada Informatics and Bio-Computing Program, Ontario Institute for Cancer Research, Toronto, Ontario M5G 0A3, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Jovelin
authors.affiliation: Department of Molecular Genetics, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Aldis
authors.initials: A
authors.lastname: Krizus
authors.affiliation: Department of Molecular Genetics, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Bakhtiyar
authors.initials: B
authors.lastname: Taghizada
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Gray
authors.affiliation: Institute of Ecology and Evolution, University of Oregon, Oregon 97403, USA.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Phillips
authors.affiliation: Department of Molecular Genetics, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Claycomb
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada.
authors.forename: Asher D
authors.initials: AD
authors.lastname: Cutter
mesh_terms: D000818:Animals; D002107:Caenorhabditis / Q000235:genetics*; D005786:Gene Expression Regulation; D035683:MicroRNAs / Q000235:genetics*; D010802:Phylogeny; D018510:RNA, Helminth / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D035683:MicroRNAs; D018510:RNA, Helminth
keywords: Caenorhabditis; cis-regulatory motifs; comparative genomics; gene regulation; miRNA