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.

Small RNAs in parasitic nematodes – forms and functions

“…Detailed studies in C. elegans and related species are beginning to reveal the mechanisms involved. Intergenic miRNAs have promoters similar to those of protein-coding genes and recent work has identified specific sequence motifs that determine expression pattern (Jovelin et al ., 2016). As gene annotation improves, the same approach, using motif discovery tool MEME, can be applied to parasitic species.…”

“…This is consistent with findings in other organisms (Liang and Li, 2009; Shen et al ., 2011) and suggests there is conservation of the machinery regulating miRNA gene expression, of which little is currently known. More recent work has identified motifs within the upstream promoter region of C. elegans miRNA genes that may determine expression level (Jovelin et al ., 2016).…”