Vol 451|24 January 2008 Q&A

MOLECULAR BIOLOGY The expanding world of small Helge Großhans and Witold Filipowicz Molecular cell biology has long been dominated by a -centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate 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 . 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 , 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 . 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- 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 , 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 (miRNAs) and Piwi-associated ery of RNAi earned its two lead researchers, have recently been identified in a unicellular 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), 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 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 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 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 , 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 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- formation. PLANT BIOL. FUNCT. as is the case for siRNAs (and possibly piRNAs), dation, facilitating ’ 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 , 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. Intuitively, terminating miRNAs were originally identified in C. elegans seems a much more obvious mechanism. But a for their central role in development. Consist- Figure 2 | miRNAs have tissue-specific block in protein production always lags behind ent with their function in differentiation and functions. In zebrafish embryos, localization a block in transcription — even if transcription development, expression of many miRNAs is patterns of individual miRNAs indicate that is stopped, mRNA sequences that have already tissue-specific (Fig. 2) or is associated with their activity could be limited to tissues and been made can still be translated into proteins. certain developmental stages. miRNA expres- organs in which they are expressed. As indicated by blue staining, miR-206 is mainly expressed So by targeting the existing mRNA pool, small sion patterns often change in diseases such as in the muscle, miR-126 in the blood vessels and RNAs block or attenuate protein synthesis very cancer. And, as many of the known and pre- the heart, miR-200a in the lateral-line system (a rapidly and, occasionally, even reversibly. In dicted miRNA targets have roles in disease, it mechanosensory system detecting water motion) addition, because individual small RNAs can is widely believed that dysregulation of miRNA and sensory organs, and miR-30c in the kidney simultaneously target tens if not hundreds of expression contributes to disease pathology. precursor.

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Box 2 | Formation and function of small RNAs For example, siRNAs spread through the vas- cular system of plants, which possibly aids their siRNA pathway miRNA pathway piRNA pathway function in antiviral immunity. And in C. ele- gans, an efficient system of siRNA amplification miRNA-encoding Viral genes ensures the maintenance of gene silencing even infection after the initial ‘trigger siRNA’ is gone, allowing siRNA to spread to the organism’s progeny. A Transcription of sense and similar amplification system does not occur in antisense strands mammals. As for miRNAs, their very specific miRNA precursor localization patterns (Fig. 2), and the absence Drosha Long, single-stranded RNA of developmental changes in C. elegans mutants dsRNA Nucleus of the dsRNA transport machinery, suggests that miRNAs are stationary. DicerCytoplasm Will small RNAs be useful as miRNA therapeutic agents or targets? duplex piRNAs siRNAs This is a hot topic of research. siRNAs have the potential to silence disease-relevant genes that cannot be shut down with available drugs. Repression of Moreover, the so-called oncomiRs — miRNAs mRNA translation miRNP piRNP RISC that promote cancer — may themselves be Piwi targets for shut-down. But we are still a long Ago Ago way from translating activity observed in a AAAA defined experimental system into an effective mRNA AAAA therapeutic drug. One of the most problem- atic issues is how to get small RNAs efficiently Germ-cell development and specifically to their target site of action in the human body. But regardless of their thera- mRNA halves mRNA fragments peutic potential, siRNAs have already revolu- tionized basic biomedical research. The use of Small RNAs are generally cellular enzymes. Under some the Argonaute (Ago) family. synthetic siRNAs, or their short hairpin RNA produced by fragmentation circumstances, siRNAs might Depending on the level of or dsRNA precursors, allows researchers to of longer precursors. associate into complexes complementarity, miRNAs repress the function of a gene of interest or Small interfering RNAs other than RISC (not shown) induce mRNA degradation or even to perform genome-wide RNAi screens (siRNAs) are processed that function in the nucleus repress their translation. Unlike to unravel entire biological pathways with from double-stranded RNAs and silence gene transcription. the siRNA pathway, miRNA- unprecedented ease and speed. (dsRNAs) that form by base- MicroRNAs (miRNAs) are mediated degradation is pairing of complementary processed from specific initiated by enzymatic removal So what of the future? RNAs. An enzyme called Dicer genome-encoded precursors, of the mRNA poly(A) tail. New classes of small RNAs continue to be dis- cleaves dsRNA into shorter which fold into intramolecular Piwi-associated RNAs covered, and it is unlikely that we have found double-stranded siRNAs that hairpins containing (piRNAs) are generated them all. Even for the known classes, we often are roughly 20 base pairs imperfectly base-paired from long, single-stranded have only a very limited understanding of long. One siRNA strand then segments. The processing precursors in a process what they do and how they do it. Identifica- assembles into an effector generally occurs in two independent of Drosha and complex known as an RNA- steps, and is catalysed by Dicer. These small RNAs tion of miRNA targets is another challenge, induced silencing complex the enzymes Drosha (in the associate with a subfamily of as is identifying other, currently hypothetical, (RISC). This complex uses nucleus) and Dicer (in the Argonaute proteins called Piwi modes of small-RNA action. Owing to their the siRNA guide to identify cytoplasm). One strand of proteins. Tens of thousands of base-pairing potential, small RNAs could mRNAs with a sequence the resulting miRNA duplex, piRNAs have been identified, modify local mRNA structures, allowing for perfectly complementary to resembling an siRNA, then although they are far from alternative mRNA splicing and modulating the siRNA. RISC then cleaves incorporates into a RISC-like understood. It is, however, interactions of mRNAs with proteins. So watch the mRNA in the middle of miRNA–ribonucleoprotein known that, together with this space. ■ the mRNA–siRNA duplex, (miRNP) complex. The main their Piwi partners, they are Helge Großhans and Witold Filipowicz are at and the resulting mRNA components of RISC and essential for the development the Friedrich Miescher Institute for Biomedical halves are degraded by other miRNPs are proteins of of germ cells. H.G. & W.F. Research, CH-4002 Basel, Switzerland. e-mails: [email protected]; [email protected] Does our collection of small RNAs set facilitate the development of new regulatory us apart from other species? relationships between these sequences, possi- FURTHER READING Some miRNAs are highly conserved, but bly contributing to the evolution of new func- Aravin, A. A, Hannon, G. J. & Brennecke, J. Science 318, 761–764 (2007). others vary greatly among organisms; some tions. Perhaps, therefore, it is not surprising Bushati, N. & Cohen, S. M. Annu. Rev. Cell. Dev. Biol. 23, differ even among primates, for example that a large fraction of tissue-specific miRNAs 175–205 (2007). between apes and . With the emerging operates in the brain. Chapman, E. J. & Carrington, J. C. Nature Rev. Genet. 8, view that regulation of protein activity could 884–896 (2007). be as vital to evolution as fidelity of the protein Are small RNAs restricted to the cells Filipowicz, W., Bhattacharyya, S. N. & Sonenberg, N. Nature Rev. Genet. 9, 102–114 (2008). sequence itself, it is tempting to speculate that in which they are made? Rana, T. M. Nature Rev. Mol. Cell Biol. 8, 23–36 (2007). miRNAs influence evolution. Relatively simple In C. elegans and plants, dsRNAs or siRNAs can Zamore, P. D. & Haley, B. Science 309, 1519–1524 requirements for miRNA–mRNA interaction move between cells or even longer distances. (2005).

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