Why do miRNAs live in the miRNP?

Abstract

Noncoding RNAs function in diverse pathways—dosage compensation, gene imprinting, transcriptional regulation, pre-mRNA splicing, and the control of mRNA translation—and they carry out these roles from within specific RNA–protein complexes that ensure each noncoding RNA is in the right cellular compartment with the appropriate proteins needed to accomplish its biochemical function. Thus, identifying the ribonucleoprotein complex (RNP) associated with a noncoding RNA gives clues to its cellular function and biochemical mechanism by revealing the proteins whose company it keeps. The discovery by Dreyfuss and coworkers that microRNAs reside in a ∼550-kD (15S) particle provides new clues toward the functions of this novel and surprisingly large class of tiny, noncoding RNAs (Mourelatos et al. 2002). The first microRNA, (miRNA) lin-4, was identified in 1993 (Lee et al. 1993). Ambros and coworkers positionally cloned the lin-4 gene, a locus required for the correct timing of development in Caenorhabditis elegans, only to find that the gene encodes no protein (Lee et al. 1993). Instead, lin-4 comprises two small noncoding RNAs, one 22 nucleotides long, and a longer form, lin-4L, that can fold into a hairpin structure. Seven years later, Ruvkun and colleagues discovered that let-7, which likewise regulates developmental timing in worms, is also a tiny, noncoding RNA (Reinhart et al. 2000). Because lin-4 and let-7 control developmental timing, they have been dubbed small temporal RNAs (stRNAs). Recently, three laboratories succeeded in cloning additional stRNA-like RNAs from worms, flies, and human cells (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). These efforts uncovered a wealth of 19–25 nucleotide RNAs, including lin-4 and let-7, which are collectively known as miRNAs (for reviews, see Moss 2001; Ruvkun 2001; Banerjee and Slack 2002). These efforts added an additional 100 tiny RNAs to the original pair of stRNAs. As anticipated by Ambros, stRNAs and miRNAs derive from longer stem-loop precursor RNAs. Thus, the longer lin-4L is the precursor of mature lin-4. Whereas many of the new miRNAs are produced constitutively, some are temporally regulated or expressed only in specific tissues. A few appear to be transcribed in coordinately regulated operons, suggesting that they are cleaved from their stem-loop precursors from within a long, common transcript. Others are found only in the germ line or in the early embryo, in which translational control dominates the hierarchy of regulatory mechanisms. The 21–25 nucleotide size of miRNAs is remarkably similar to that of small interfering RNAs (siRNAs), the 21–25 nucleotide double-stranded RNAs that mediate RNA interference (for reviews, see Bernstein et al. 2001b; Carthew 2001; Sharp 2001; Vaucheret et al. 2001; Waterhouse et al. 2001). siRNAs are generated by the endonucleolytic cleavage of long double-stranded RNA by the multidomain RNase III enzyme, Dicer (Bernstein et al. 2001a). siRNAs are then incorporated into a ∼500-kD RNP complex, the RNA-induced silencing complex (RISC), in which they provide the specificity determinants that direct an as yet unidentified protein nuclease to cleave mRNAs complementary to the siRNA (Hammond et al. 2000). lin-4 and let-7, as well as the new miRNAs, are encoded by ∼70 nucleotide stem-loop structures (Lee et al. 1993; Pasquinelli et al. 2000), whose stems are substrates for processing by Dicer (Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001). Dicer liberates miRNAs from the larger stem-loop precursors in much the same way it generates siRNAs from long dsRNA, leaving the signature 3 hydroxyl and 5 phosphate termini of an RNase III cleavage reaction. Both siRNA and stRNA production by Dicer requires ATP, consistent with the presence of an ATP-dependent helicase domain at the amino terminus of Dicer (Zamore et al. 2000; Bernstein et al. 2001a; Hutvagner et al. 2001; Nykanen et al. 2001). Mature lin-4 and let-7 are thought to bind partially complementary sequences in the 3 UTRs of their target mRNAs (Lee et al. 1993; Reinhart et al. 2000). Unlike the binding of siRNAs, which triggers target RNA destruction, binding of the stRNA lin-4, and likely let-7, leads to translational repression of their natural mRNA targets (Olsen and Ambros 1999; Reinhart et al. 2000; Slack et al. 2000). In worms, translational repression of lin-4 and let-7 target mRNAs is required for the progression from one stage of development to the next. In addition to Dicer, two members of the PPD family of proteins, ALG-1 and ALG-2, are required for the bio1Corresponding author. E-MAIL phillip.zamore@umassmed.edu; FAX (508) 856-2003. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.992502.