In FOCUS Regulate Biology

Rachel Green†,‡,* and Jennifer A. Doudna†,§,* †Howard Hughes Medical Institute and ‡Department of and Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, and §Department of Molecular and Cell Biology and Department of Chemistry, University of California, Berkeley, California 94720

e both entered the RNA field in Rather than attempt to summarize every the late 1980s, when catalytic topic covered at the meeting (a nearly W RNAs and in vitro selection impossible task!), this review focuses on approaches were an exciting new frontier. several of the key themes that emerged from The first scientific meeting one of us (J.A.D.) the many presentations and informal dis- attended was the 52nd Cold Spring Harbor cussions that occurred during the course of Symposium (CSHS) on the Evolution of Cata- the symposium. lysts in the spring of 1987. It was a spec- A central topic was the discovery of new tacular experience: attendees at the noncoding RNAs (ncRNAs). What’s out meeting were abuzz with the excitement there? The resounding answer: a lot! ncRNAs and novelty of ribozymes and the mecha- are abundant in all three kingdoms of life, as nisms of chemical reactions critical for life. revealed by a myriad of approaches, includ- Ample time to mingle with colleagues fos- ing direct cloning and sequencing of cellular tered many conversations about the details RNA, computational prediction, and micro- and unanticipated connections among the array analysis. Several research groups are systems we were each studying. using sequencing methodology, referred to Almost 20 years later, the world of func- as 454 (www.454.com), for the “deep tional RNAs is no less exciting. This year’s sequencing” of many thousands of small 71st CSHS on Regulatory RNAs, held RNAs (sRNAs). This technique has enabled May 31–June 5, 2006, in Cold Spring the rapid compilation of large numbers of Harbor, NY, discussed novel aspects of RNA sequences that can be sorted by size, biology that we are only beginning to under- sequence, evolutionary conservation, and stand. Focused largely, though not exclu- location within a genome. sively, on RNA interference (RNAi), the As a result, new classes of small ncRNAs meeting brought together a diverse group of are rapidly being identified, although the scientists broadly interested in understand- functions for these molecules remain myste- ing how, where, when, and why RNA mol- rious. What is clear so far is that size ecules have evolved to regulate matters. In plants, for example, David Baul- expression in a wide variety of ways in cells combe described how four distinct variants and viruses. Although the pathways and of the RNA-cleaving enzyme Dicer each molecular players involved in RNA-mediated produce double-stranded RNA (dsRNA) *Corresponding authors, gene regulation are being elucidated at a products (micro- or small interfering RNAs) [email protected], [email protected]. rapid pace, the chemical and mechanistic of a characteristic length that confer on underpinnings remain to be worked out. The them the ability to enter particular gene- underlying molecular mechanisms, and the regulating pathways. Steve Jacobsen and possibilities for tapping these processes for Richard Jorgensen presented genetic Published online July 21, 2006 therapeutic purposes, fall squarely into the studies that similarly emphasized the diver- 10.1021/cb600277m CCC: $33.50 realm of chemical biology. sity of RNAi-mediated silencing pathways © 2006 by American Chemical Society www.acschemicalbiology.org VOL.1 NO.6 • ACS CHEMICAL BIOLOGY 335 that appear to function in different rapid clip, including recent crystal plants. Greg Hannon (1) presented structures of the guanine (4) and a large family of ϳ30-mer RNAs, S-adenosylmethionine-binding found exclusively in murine testes, riboswitches (5) presented by that have been dubbed piRNAs Robert Batey and a structure of the because of their propensity to bind E. coli thiamine pyrophosphate- Piwi-domain-containing in binding riboswitch presented on a these germ cells. These 30-mers, poster from Dinshaw Patel’s lab which usually have a 5= uridine (6). The remarkable T-box RNA residue, are typically encoded in © Miriam Chua, 1999 described by Tina Henkin recog- intergenic regions clustered in the genome nizes specific transfer RNAs (tRNAs) and and are not conserved at the sequence level formed in cell culture. Other intriguing con- changes structure depending on whether the in other organisms. Phil Zamore presented nections among RNA editing, transport, and tRNA is charged with its cognate amino acids; data suggesting that a similar class of RNAi were touched upon in talks by Brenda thus, expression of bacterial in sRNAs, the rasiRNAs (repeat-associated Bass and Gordon Carmichael, highlighting response to cells’ nutritional status is con- silencing RNAs), might be generated the extensive interplay among these seem- trolled (7). One wonders whether such rela- through a previously undescribed pathway. ingly distinct processes. Tom Gingeras and tively small RNAs, linked together, could In the nematode Caenorhabditis elegans, Mike Snyder described results from the function as a primitive if supplied David Bartel has found that almost one- ENCODE (Encyclopedia of DNA Elements) with an mRNA template, food for thought tenth of the sequences identified so far by project aimed at characterizing all the tran- about the evolutionary origins of RNA- the 454 method corresponds to so-called scripts produced from 1% of the human catalyzed biosynthesis. 21U-RNAs. These are 21-mer RNAs that genome. Findings from their labs and others Many of the identified sRNAs whose func- always contain a 5= uridine residue and are suggest that much more of the genome is tions are known are microRNAs (miRNAs), derived from thousands of loci in two broad transcribed than previously known, at least and hence, numerous labs have used regions of chromosome IV, dispersed at a low level, and more than half of all genetic and biochemical approaches to set between protein-coding genes and within coding genes have very distal, alternative, about finding their molecular partners in their introns. No precise function for piRNAs tissue-specific transcription start sites. Nick different organisms. Scientists from the or 21U-RNAs has yet been determined. Proudfoot presented evidence that aberrant, Ruvkun and Carthew labs described nega- ncRNAs are not always small. RNAs pro- intergenic, and genic globin locus tran- tive regulators of RNAi in nematodes and duced by RNA polymerase II, the same scripts are subjected to RNAi mechanisms, fruit flies; that cells might control RNAi in enzyme that transcribes precursor messen- whereas regular globin transcripts are made response to environmental stimuli was sug- ger RNAs (mRNAs) in the nucleus, are some- from genes arranged in transcription-depen- gested. Craig Hunter presented evidence for times retained in the nucleus rather than dent loop structures. a transmembrane transporter of dsRNA exported to sites of protein synthesis in the ncRNAs are not unique to : required for the spreading of RNA silencing cytoplasm. Two fascinating examples dis- also contain small regulatory RNAs between cells in nematodes (8, 9). cussed by David Spector include the 9-kb (so far at least 80 have been identified in Crystallographic structure determinations CTN-RNA and another 7-kb RNA, found in ), and use structured RNAs of RNAi pathway components, presented by murine and human cells. The CTN-RNA called riboswitches (as described by Wade the Barford (10), Joshua-Tor (11), and appears to be a long version of an mRNA Winkler, Tina Henkin, and Eduardo Grois- Doudna (12) and Patel labs (13, 14), show encoding an transporter, raising man) to control gene expression in response how siRNAs are likely to bind within com- the possibility that CTN-RNA is a storage to a wide array of small molecules, including plexes containing Dicer and the mRNA form of the message that is on hand for magnesium ions (2). Gigi Storz shared new target-cleaving endonuclease Argonaute 2. rapid processing and export in the event of data indicating that small bacterial RNAs These structures are guiding biochemical amino acid starvation. The 7-kb-long RNA is can mediate their effects by binding to the investigation of the molecular mechanisms also found in the nucleus, in this case in 3= untranslated region of the gene, reminis- that underlie the individual steps in RNAi. neurons at sites of preliminary mRNA pro- cent of many examples of translational How miRNAs work in vivo remains a sub- cessing, and its abundance intriguingly cor- control in eukaryotes (3). Molecular struc- ject of much discussion and debate. Al- relates with the numbers of neural synapses tures of riboswitches are appearing at a though evidence in some experimental

336 VOL.1 NO.6 • 335–338 • 2006 GREEN AND DOUDNA www.acschemicalbiology.org In FOCUS

systems and from computational approaches of these points home. She showed that mul- mosomes in certain cancerous human cells. suggests that miRNAs can fine-tune gene tiple pathways exist for regulation and that Data from the Cech lab suggest that regulation, data from the Hobert lab show simply knowing that a gene is controlled by telomerase RNA, which includes the tem- that a miRNA in the nematode nervous a miRNA does not reveal how it is controlled. plate sequence for extending the telomeric system is a clear switch that determines left- Peter Sarnow’s talk about a hepatic miRNA stretches at the ends of the chromosomes, versus right-brain functional asymmetry (15, established that miRNAs can function as is a flexible scaffold for assembling telomer- 16). These studies are consistent with early activators rather than as repressors (20). ase proteins whose copy number is impor- genetic observations in the field and sug- The take-home message is to keep an open tant for proper telomere maintenance gest the possibility that such on–off control mind in examining every new case: the rules in vivo. Carol Greider presented a genetic of gene expression by miRNAs will be more are far from established. story indicating that half the amount of generally observed. Although miRNA tissue Discussed at the meeting was transcrip- telomerase RNA over multiple generations localization can be exquisitely specific, as tional silencing, another broad topic that results in a heritable phenotype associated shown in the zebrafish by Ron Plasterk, just incorporates both small and large RNAs and with shortened telomeres (26, 27). An excit- a quarter of the miRNAs for which chemi- their cellular effects. Danesh Moazed and ing talk on alternative splicing from Bob cally stable antisense oligonucleotides were Shiv Grewal presented complementary Darnell provided new insight into how RNP used to block function had a discernible stories on the idea of a self-reinforcing loop complexes regulate which parts of a pre- phenotype. That said, Frank Slack shared of processes responsible for sRNA-mediated mRNA are stitched together to produce compelling evidence for the involvement of gene silencing: transcription makes RNAs, tissue-specific messages. Gideon Drey- the let-7 miRNA family in regulating the RNA-dependent RNA polymerase amplifies fuss’s dissection of the SMN (survival of expression of important oncogenes impli- them, Dicer processes, Argonautes bind, motor neurons) complex is beginning to cated in lung cancer. Alex Schier showed an and assembled RNA-induced transcriptional reveal some of the biochemical rules for example of another way to affect gene silencing complexes act in cis at the locus splicing complex assembly (28, 29). expression, whereby a miRNA promotes to degrade nascent transcripts (21–24). After a dizzying week thinking about deadenylation of maternal mRNAs during Although initially surprising, transcriptional mechanisms in a seemingly overwhelming early embryogenesis (17, 18). In a related silencing depends on transcription of the sea of connections in RNA biology, we will story, Jim Dahlberg and Elsebet Lund locus. Edith Heard spoke about how specific all remember the striking stories that we reported that miRNA biogenesis is con- subnuclear zones appear to be established heard that bring home just how central trolled during Xenopus laevis egg maturation for gene silencing by Xist RNA, and Jeannie these processes are to understanding life. and that miRNA-dependent deadenylation Lee discussed how direct physical pairing Surely one of the most memorable talks can occur in the absence of . between the X chromosomes is important about biology was by Michel Georges, who One particularly vexing question is how for establishing chromosome inactivation studies why Texel sheep are “exceptionally miRNAs are able to down-regulate protein (25). As we have learned for miRNA- meaty”; the answer appears to be that these synthesis without affecting mRNA levels, a mediated gene regulation, localization is a sheep carry a mutation in the untranslated process called translational repression. big factor in explaining how these processes region of the myostatin gene that creates a Several speakers, including Witold Filipo- take place. As biochemists, we should note binding site for an already expressed miRNA wicz, Phil Sharp, and Tim Nilsen, presented that biology depends on localization; (30). We are only beginning to appreciate biochemical and cell biological studies that although we often may be able to mimic how much of known phenotypic variation attempted to decipher the mechanism(s) such effects in vitro with high concentra- can be explained by these novel classes of of gene regulation by miRNAs (19). And, tions, we may not always be successful. regulators, the sRNAs. although little consensus exists about what With so much of the meeting focused on stage of translation (or some other process) RNA, it was refreshing to hear several pre- is being controlled by miRNA interactions, sentations about RNPs, ribonucleoprotein REFERENCES all would agree that determining where the complexes that facilitate the functional 1. Girard, A., Sachidanandam, R., Hannon, G. J., and Carmell, M. A. (2006) A germline-specific class of components are localized would provide association of RNAs with their in vivo protein small RNAs binds mammalian Piwi proteins, Nature, important insight and that processing partners. In a session focused on telomer- published online June 4, http://dx.doi.org/ bodies (P bodies) and stress granules figure ase and cancer, Liz Blackburn presented her 10.1038/nature04917. 2. Cromie, M. J., Shi, Y., Latifi, T., and Groisman, E. A. prominently in these discussions. 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