Molecular characterization of animal microRNAs : sequence, expression, and stability

Abstract

(cont.) miRNAs. These cells lines may also be useful for other functional studies, such as validation of putative mRNA target genes.

Multicellular organisms possess natural gene-regulatory pathways that employ small RNAs to negatively regulate gene expression. In nematodes, the small temporal RNAs (stRNAs), lin-4 and let-7, negatively regulate genes important in specifying developmental timing. A gene-silencing pathway present in plants, fungi and animals called RNA interference, involves the conversion of long double-stranded RNA into short interfering RNAs, which can serve to negatively regulate endogenous genes or suppress the replication of viruses and transposons. To investigate how wide a role small RNAs play in regulating gene expression in animals, we developed a RNA cloning procedure and first applied it to the cloning of small RNAs from the nematode, Caenorhabditis elegans. In addition to cloning lin-4 and let-7 sequences, our study revealed a large number of conserved and highly expressed small RNAs with features reminiscent of stRNAs. Because not all of these small RNAs were expressed in temporal fashion, we and others have referred to this novel class of tiny RNAs as microRNAs. We completed an extensive census of microRNA (miRNA) genes in C.elegans by cloning and bioinformatics searches to lay the groundwork for future functional studies. Our census marked the detection of nearly 90 C.elegans miRNAs, estimated an upper-bound of about 120 miRNA genes in C.elegans, and detailed the conservation and clustering of miRNA sequences. We also determined the high molecular abundance of several miRNAs in C.elegans and Hela cells. In an effort to understand the reason for the high molecular abundance of miRNAs, we constructed an inducible miRNA-expressing cell line to measure the stability of animal miRNAs. Time course measurements suggested a long (>24 hours) half-life for two