Content area
Full text
In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.
Two distinct pathways exist in animals and plants in which 21- to 23-nt RNAs function as posttranscriptional regulators of gene expression. Small interfering RNAs (siRNAs) act as mediators of sequence-specific mRNA degradation in RNA interference (RNAi) (15), whereas stRNAs regulate developmental timing by mediating sequence-specific repression of mRNA translation (6-11). siRNAs and stRNAs are excised from double-stranded RNA (dsRNA) precursors by Dicer (12-14), a multidomain ribonuclease III protein, thus producing RNA species of similar sizes. However, siRNAs are believed to be double-stranded (2, 5, 12), whereas stRNAs are single-stranded (8).
We previously developed a directional cloning procedure to isolate siRNAs after processing of long dsRNAs in Drosophila melanogaster embryo lysate (2). Briefly, 5' and 3' adapter molecules were ligated to the ends of a size-fractionated RNA population, followed by reverse transcription polymerase chain reaction (PCR) amplification, concatamerization, cloning, and sequencing. This method, originally intended to isolate siRNAs, led to the simultaneous identification of 16 novel 20- to 23-nt short RNAs, which are encoded in the D. melanogaster genome and are expressed in 0- to 2-hour embryos (Table 1). The method was adapted to clone RNAs in a similar size range from HeLa cell total RNA (15), which led to the identification of 21 novel human microRNAs (Table 2), thus providing further evidence for the existence of a large class of small RNAs with potential regulatory roles. Because of their small size, and in agreement with the authors of two related papers in this issue (16, 17), we refer to these novel RNAs as microRNAs (miRNAs). The miRNAs we studied are abbreviated as miR-1 to miR-33, and the genes encoding miRNAs are named mir-1 to mir-33. Highly homologous miRNAs are referred to by the same gene...