Why Do Mirnas Live in the Mirnp?
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Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Why do miRNAs live in the miRNP? Dianne S. Schwarz and Phillip D. Zamore1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA Do tiny RNAs now have a home? the new miRNAs are produced constitutively, some are temporally regulated or expressed only in specific tis- Noncoding RNAs function in diverse pathways—dosage sues. A few appear to be transcribed in coordinately regu- compensation, gene imprinting, transcriptional regula- lated operons, suggesting that they are cleaved from their tion, pre-mRNA splicing, and the control of mRNA stem-loop precursors from within a long, common tran- translation—and they carry out these roles from within script. Others are found only in the germ line or in the specific RNA–protein complexes that ensure each non- early embryo, in which translational control dominates coding RNA is in the right cellular compartment with the hierarchy of regulatory mechanisms. the appropriate proteins needed to accomplish its bio- The 21–25 nucleotide size of miRNAs is remarkably chemical function. Thus, identifying the ribonucleopro- similar to that of small interfering RNAs (siRNAs), the tein complex (RNP) associated with a noncoding RNA 21–25 nucleotide double-stranded RNAs that mediate gives clues to its cellular function and biochemical RNA interference (for reviews, see Bernstein et al. 2001b; mechanism by revealing the proteins whose company it Carthew 2001; Sharp 2001; Vaucheret et al. 2001; Water- keeps. The discovery by Dreyfuss and coworkers that house et al. 2001). siRNAs are generated by the endonu- microRNAs reside in a ∼550-kD (15S) particle provides cleolytic cleavage of long double-stranded RNA by the new clues toward the functions of this novel and surpris- multidomain RNase III enzyme, Dicer (Bernstein et al. ingly large class of tiny, noncoding RNAs (Mourelatos et 2001a). siRNAs are then incorporated into a ∼500-kD al. 2002). RNP complex, the RNA-induced silencing complex The first microRNA, (miRNA) lin-4, was identified in (RISC), in which they provide the specificity determi- 1993 (Lee et al. 1993). Ambros and coworkers position- nants that direct an as yet unidentified protein nuclease ally cloned the lin-4 gene, a locus required for the correct to cleave mRNAs complementary to the siRNA (Ham- timing of development in Caenorhabditis elegans, only mond et al. 2000). lin-4 and let-7, as well as the new to find that the gene encodes no protein (Lee et al. 1993). miRNAs, are encoded by ∼70 nucleotide stem-loop Instead, lin-4 comprises two small noncoding RNAs, one structures (Lee et al. 1993; Pasquinelli et al. 2000), whose 22 nucleotides long, and a longer form, lin-4L, that can stems are substrates for processing by Dicer (Grishok et fold into a hairpin structure. Seven years later, Ruvkun al. 2001; Hutvágner et al. 2001; Ketting et al. 2001). Dicer and colleagues discovered that let-7, which likewise liberates miRNAs from the larger stem-loop precursors regulates developmental timing in worms, is also a tiny, in much the same way it generates siRNAs from long noncoding RNA (Reinhart et al. 2000). Because lin-4 and dsRNA, leaving the signature 3Ј hydroxyl and 5Ј phos- let-7 control developmental timing, they have been phate termini of an RNase III cleavage reaction. Both dubbed small temporal RNAs (stRNAs). Recently, three siRNA and stRNA production by Dicer requires ATP, laboratories succeeded in cloning additional stRNA-like consistent with the presence of an ATP-dependent heli- RNAs from worms, flies, and human cells (Lagos-Quin- case domain at the amino terminus of Dicer (Zamore et tana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). al. 2000; Bernstein et al. 2001a; Hutvágner et al. 2001; These efforts uncovered a wealth of 19–25 nucleotide Nykänen et al. 2001). Mature lin-4 and let-7 are thought RNAs, including lin-4 and let-7, which are collectively to bind partially complementary sequences in the 3Ј known as miRNAs (for reviews, see Moss 2001; Ruvkun UTRs of their target mRNAs (Lee et al. 1993; Reinhart et 2001; Banerjee and Slack 2002). These efforts added an al. 2000). Unlike the binding of siRNAs, which triggers additional 100 tiny RNAs to the original pair of stRNAs. target RNA destruction, binding of the stRNA lin-4, and As anticipated by Ambros, stRNAs and miRNAs derive likely let-7, leads to translational repression of their from longer stem-loop precursor RNAs. Thus, the longer natural mRNA targets (Olsen and Ambros 1999; Rein- lin-4L is the precursor of mature lin-4. Whereas many of hart et al. 2000; Slack et al. 2000). In worms, transla- tional repression of lin-4 and let-7 target mRNAs is re- quired for the progression from one stage of development 1Corresponding author. to the next. E-MAIL [email protected]; FAX (508) 856-2003. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ In addition to Dicer, two members of the PPD family gad.992502. of proteins, ALG-1 and ALG-2, are required for the bio- GENES & DEVELOPMENT 16:1025–1031 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 1025 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Schwarz and Zamore genesis or function of lin-4 and let-7 in worms (Grishok (Knight and Bass 2001). Dreyfus and coworkers now find et al. 2001). PPD proteins, so named because they con- that many human miRNAs are present in a ∼550-kD tain PAZ and Piwi domains, protein sequence motifs of complex, the miRNP, and that this complex contains the unknown biochemical function, are required for a diverse PPD protein eIF2C2 (Mourelatos et al. 2002). array of developmental functions in plants and animals. Current evidence supports the view that in metazoans, alg-1/alg-2 mutants accumulate lin-4 and let-7 precur- both the RNAi and miRNA pathways require the activ- sors and display striking defects in developmental tim- ity of Dicer and PPD proteins. The RNAi and miRNA ing (Grishok et al. 2001). Worms lacking alg-2 also fail to pathways are clearly related, but there are features that form a normal germ line (Cikaluk et al. 1999). A role for differentiate them. First, siRNAs are processed from per- PPD proteins in the biogenesis or function of miRNAs fectly complementary, long dsRNA into double-stranded has only been shown for the PPD proteins ALG-1 and siRNAs that guide the destruction of a target mRNA ALG-2 in worms, but it seems likely that PPD proteins (Hamilton and Baulcombe 1999; Hammond et al. 2000; will be needed for miRNA biogenesis in other organisms. Zamore et al. 2000; Bernstein et al. 2001a; Elbashir et al. PPD proteins function not only in miRNA matura- 2001a,b; Nykänen et al. 2001). In contrast, miRNAs are tion, but are also required in animals, plants, and fungi single stranded, processed from ∼70 nucleotide precur- for a variety of RNA-silencing phenomena, including sors that have the ability to form stem-loop structures RNAi and cosuppression, the RNAi-like silencing of an containing loops and bulges of unpaired nucleotides. endogenous gene by a transgene copy of the same se- Only one strand of the stem of the miRNA precursor quence (Tabara et al. 1999; Catalanotto et al. 2000; Fa- accumulates, indicating that the other strand must ei- gard et al. 2000; Hammond et al. 2001; Pal-Bhadra et al. ther not be produced or is differentially degraded. The 2002). In C. elegans, the PPD protein RDE-1 is required fact that multiple PPD proteins are found in various or- for RNAi (but not cosuppression; Tabara et al. 1999; ganisms that exhibit RNAi may be an indication that Dernburg et al. 2000; Ketting and Plasterk 2000); QDE-2 different classes of these proteins play a specialized role is required for cosuppression in the fungus Neurospora in the two pathways. But reality must be more complex crassa (Catalanotto et al. 2000); and Argonaute is re- than this simple model, which predicts that two PPD quired both for RNA silencing and for normal meristem proteins—one for RNAi and one for miRNAs—would function in plants (Bohmert et al. 1998; Fagard et al. suffice. Instead, the number of PPD proteins is large and 2000). In flies, the PPD protein, Piwi, is required for the varies greatly in different organisms (Fig. 1): flies have maintenance of germ-line stem cells, for the post-tran- five, humans have four, and Arabidopsis have six, but scriptional silencing of endogenous genes by transgenes worms have at least 24! encoding the same mRNA, and even for some aspects of transcriptional silencing (Cox et al. 1998; Pal-Bhadra et Why so many PPD proteins? al. 2002). Intriguingly, Piwi localizes to the nucleoplasm, not the cytoplasm, in Drosophila ovaries and testes, but One explanation is that not all PPD proteins function in disperses to the cytoplasm during mitosis (Cox et al. the RNAi or miRNA pathways. This seems unlikely, as 2000). The role of Piwi in post-transcriptional silenc- three of the five Drosophila family members have al- ing—a phenomenon that all current evidence suggests ready been implicated in one or another RNA-silencing occurs in the cytoplasm—implies that at least a sub- phenomenon. Perhaps different subclasses of miRNAs population of Piwi functions outside of the nucleus. require distinct PPD proteins for their production, sta- Might Piwi associate with miRNAs in the cytoplasm, bility, or function.