Vol 451|24 January 2008 Q&A MOLECULAR BIOLOGY The expanding world of small RNAs Helge Großhans and Witold Filipowicz Molecular cell biology has long been dominated by a protein-centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate gene expression at different levels, and have essential roles in health and disease. How many classes of RNA have for inactivation by classical genetic tools. More- per cent of protein-coding genes. Moreover, been identified? over, many miRNAs can compensate for each new classes of small RNAs continue to be There are three main types of ‘classic’ RNA: other’s function, which makes their identifica- discovered. messenger RNA, transfer RNA and ribosomal tion on the basis of the overt consequences of RNA. mRNAs are translated into proteins, their absence difficult. The first two miRNAs Do all organisms have small RNAs? whereas tRNAs and rRNAs have housekeeping to be discovered, lin-4 and let-7, were identified With the notable exception of the budding yeast roles during mRNA translation. Small RNAs in the 1990s through genetic experiments in Saccharomyces cerevisiae, almost all eukaryo- are not translated into proteins. Instead, these the worm Caenorhabditis elegans. Then siRNAs tic organisms (the cells of which contain a 20–30-nucleotide sequences regulate vari- were identified in animals, plants and fungi as nucleus) investigated so far have siRNAs or ous biological processes, often by interfering the effector molecules that mediate the pro cess at least cellular machineries to produce them. with mRNA translation. Small RNAs come in of sequence-specific gene silencing, or RNA Evolutionarily, miRNAs seem to be younger different forms (Box 1), the best-understood interference (RNAi), in response to double- than siRNAs. miRNAs function primarily classes being small interfering RNAs (siRNAs), stranded RNA (dsRNA; Fig. 1). The discov- in multicellular organisms, although they microRNAs (miRNAs) and Piwi-associated ery of RNAi earned its two lead researchers, have recently been identified in a uni cellular RNAs (piRNAs). Andrew Fire and Craig Mello, the 2006 Nobel alga, Chlamydomonas reinhardtii. Certain Prize in Physiology or Medicine. DNA viruses also express miRNAs. Although When were small RNAs discovered? 20–30-nucleotide small RNAs have not been During the past two to three decades, Have we identified all small RNAs? identified in archaea and eubacteria (the cells researchers identified many small regula- Hardly. Although hundreds of small RNAs of which lack a nucleus), Argonaute proteins tory RNAs, ranging in size from roughly 70 have been identified in plants and animals, — the effector molecules of small RNAs — are to 300 nucleo tides, in various organisms. But high-throughput sequencing and sophisti- present in some of these organisms. the 20–30-nucleotide small regulatory RNAs cated bioinformatics tools repeatedly reveal the were discovered more recently because, owing existence of new miRNAs and siRNAs. The How are small RNAs made? to their small size, they are easily missed in human genome could encode more than a Generally, by fragmentation of longer RNA biochemical analysis, and they are poor targets thousand miRNAs, the equivalent of a few sequences. The precursors of miRNAs and siRNAs are dsRNAs, which are processed to Box 1 | Prominent members of the RNA family small RNAs by dedicated sets of enzymes and Classic RNAs mediating Non-coding regulatory RNAs plants and animals. They are other proteins (Box 2, page 416). protein synthesis siRNAs (small interfering processed from long, single- mRNAs (messenger RNAs) RNAs) Small RNAs (20–25 stranded RNA sequences that Where do the dsRNAs come from? Transcripts of protein-coding nucleotides in length) fold into hairpin structures. Depending on the class of small RNA, the genes that act as templates for formed through cleavage piRNAs (Piwi-associated source of precursor dsRNA differs. For protein synthesis. of long double-stranded RNAs) Small RNAs (25–30 siRNAs, dsRNA can form when comple- rRNAs (ribosomal RNAs) RNA molecules. siRNAs nucleotides in length) that mentary DNA strands are transcribed into RNA constituents of are particularly important are generated from long RNA sequences. Viral infection of a cell can the ribonucleoprotein for taming the activity of single-stranded precursors. also supply dsRNAs, as many viruses form particles known as transposons and combating They function in association RNAs of both sense and antisense polar- ribosomes, which mediate viral infection, but they can with the Piwi subfamily of ity during replication of their genomes, and the decoding of mRNAs to also regulate protein-coding Argonaute proteins, and are this can trigger an RNAi response by the cell, the amino-acid sequences genes. Synthetic siRNAs can essential for the development as part of its antiviral defence. By contrast, of proteins. also be artificially expressed of germ cells. miRNAs are excised from ‘purpose-built’, tRNAs (transfer RNAs) for experimental purposes. Longer non-coding RNAs genome-encoded RNA precursors that Adapter molecules miRNAs (microRNAs) Small RNAs of 70 to thousands fold into long hairpins resembling dsRNA. carrying individual amino RNAs (20–25 nucleotides in of nucleotides that acids to the site of protein length) that are encoded by participate in various cellular The expression of miRNA-encoding genes synthesis that recognize specific genes and function in processes, including mRNA and those encoding mRNAs are controlled specific codons in repressing mRNA translation splicing and ribosome very similarly and involve the same RNA- mRNA. or in mRNA degradation in biogenesis. H.G. & W.F. synthetic machinery, including the enzyme RNA polymerase II. 414 NATURE|Vol 451|24 January 2008 NEWS & VIEWS Q&A 279–289 (1990) 279–289 2, PLANT CELL 401–409 (1998); 13, ALL IMAGES R. JORGENSEN, UNIV. ARIZONA, TUCSON ARIZONA, UNIV. R. JORGENSEN, ALL IMAGES Wild type PLANT J. RNAi Figure 1 | First phenotypic description of RNA interference. White sections in petunia flowers represent areas where RNAi has silenced a gene involved in flower coloration. How do small RNAs function? mRNAs for different proteins, they are well It is less clear whether siRNAs have similarly (2002); 1501–1506 29, They recognize their RNA targets by sequence- suited to coordinate the expression of genes important functions, although in plants they specific base-pairing. The outcome of the small- that function in the same or related pathways. have already been identified as essential play- RNA–mRNA association depends on the degree For example, during zebrafish embryonic ers in the regulation of stress resistance. In of complementarity between the two sequences. development, a specific miRNA, miR-430, fission yeast and plants, siRNAs contribute to When base-pairing is perfect, or almost perfect, targets hundreds of mRNAs for rapid degra- heterochromatin formation. PLANT BIOL. FUNCT. as is the case for siRNAs (and possibly piRNAs), dation, facilitating embryos’ transition to a the target mRNA is cleaved in the middle of the new developmental programme that requires And what is the link to viruses? small-RNA–mRNA duplex. Most plant miRNAs a separate set of proteins. The ability of dif- The use of RNAi as a defence mechanism and some animal miRNAs function similarly. ferent miRNAs to concurrently target several against viruses may have been a driving force But most animal miRNAs base-pair imprecisely sequences of the same mRNA further increases in the evolution of the siRNA pathway. In RIGHT: LEFT TO FROM with mRNAs to repress their translation or to their potential to fine-tune gene expression. plants, siRNAs are an essential layer of anti- induce their breakdown. Irrespective of base- viral defence. Also, in plants and invertebrates, pairing precision, small RNAs rely on proteins Is all of this small-RNA-mediated siRNAs silence mobile genetic elements called of the Argonaute family for their activity. In fact, regulation post-transcriptional? transposons, which would otherwise ‘jump’ it is the protein partners of small RNAs that No — small RNAs also affect DNA transcrip- around the genome and disrupt cellular genes. bring about repression of translation or mRNA tion, particularly in plants and fission yeast. It is not well known whether these small- cleavage; small RNAs act only as guides to tell They do this by sequence-specific targeting of RNA functions are also crucial in vertebrates, Argonaute proteins which mRNAs to target. chromatin (complexes of DNA with histone in which the invention of a protein-based proteins), converting it to the hetero chromatin adaptive immune response may have reduced So can their mRNA targets be predicted form that is not easily accessible to the trans- reliance on antiviral RNAi activity. through sequence analysis? criptional machinery. Strikingly, in some Unfortunately, the devil is in the detail — at lower eukaryotes small RNAs also direct least in the case of animal miRNAs, which massive genomic DNA rearrangements. In mostly base-pair to their mRNA targets with mammals, however, there is currently only limited complementarity. Although sequence limited evidence for small-RNA functions miR-206 310–311 (2005) 309, analyses have revealed some criteria for inter- other than post-transcriptional regulation. action between miRNA and mRNA, and many SCIENCE bioinformatics tools for target identification Do small RNAs always silence are available, sequence-based predictions gene expression? ET AL. miR-126 frequently yield false positives or miss true In some conditions, small RNAs may also targets. So identification of bona fide miRNA activate gene expression, although the mech- targets requires extensive experimentation. anisms are currently not well understood. By contrast, most targets of siRNAs and plant Indeed, a liver-specific miRNA, miR-122, is E. WIENHOLDS miRNAs can be reliably predicted on the basis even needed for successful replication of the miR-200a of near-perfect sequence complementarity. hepatitis C virus. To prevent protein synthesis, isn’t it What biological processes do simpler to stop mRNA production? small RNAs regulate? miR-30c Yes.