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It’s a Small RNA World, After All Matthew W. Vaughn and Rob Martienssen Small RNAs (sRNAs) can regulate transcript and protein abundance. Previously, they have been identified using traditional cloning approaches, which has limited how many could be characterized. Now, the Meyers and Green laboratories have used massively parallel signature sequencing technology to find over 1.5 million sRNAs in Arabidopsis thaliana. These new sRNAs reveal a greater-than-expected potential role for sRNAs in gene regulation, preferential expression or usage of sRNAs in flowers, and the prospect of targeted sRNA-mediated regulation of pseudogenes. In addition, new plant microRNAs have been identified, some of which may be unique to Arabidopsis. Small RNAs (sRNAs) are 20- to 26-nucleotide noncoding RNAs that regulate transcript and protein abundance via multiple mechanisms (1, 2). MicroRNAs (miRNAs) are generated by endonucleolytic cleavage of hairpin precursor transcripts by Dicer ribonuclease (RNase) III–like proteins and can direct the cleavage of target transcripts by Argonaute RNAse H–like proteins in a sequence-specific manner. miRNAs can also inhibit translation of target mRNAs. Small interfering RNAs (siRNAs) are generated by Dicer-mediated processive cleavage of double-stranded RNA (dsRNA). They can direct cleavage of other transcripts and can also promote second-strand synthesis by RNAdependent RNA polymerase (RdRP), resulting in dsRNAs. In addition, siRNAs are implicated in recruiting heterochromatic modifications that result in transcriptional silencing. Previously, sRNAs have been identified by means of painstaking cloning and sequencing techniques, and as a result, only a few thousand Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. have been identified. A recent study from the laboratories of Green and Meyers (3) describes the use of massively parallel signature sequencing (MPSS) technology to identify over 1.5 million sRNAs from Arabidopsis thaliana, representing over 75,000 distinct sequences. They report that many more genes may be under the control of sRNAs than had been previously imagined. In Arabidopsis, current estimates predict that 2% of genes may be under miRNA control (4), but the number of genes that might be regulated by siRNAs is not known. siRNAs are known to participate in RNAi-mediated silencing of repeats and transposable elements (TEs) (5), so it was no surprise that the majority of sRNA sequences matched repeats and that most annotated repeats and TEs matched abundant sRNAs (3). Most of the remaining sRNA sequences came from intergenic regions (IGRs), including those derived from miRNAs, which is consistent with previous studies on a much smaller scale (6). Interestingly, 4000 protein-coding genes (15% of genes in the genome) and several hundred RNA VIEWPO<strong>IN</strong>T pseudogenes matched at least one sRNA perfectly, presumably corresponding to siRNAs (miRNAs usually match imperfectly). However, most of these genes matched only one siRNA, and only a few percent of the total sRNA sequences were from genes. It is possible therefore that many other genes may have matches that went undetected. The number of distinct sRNA sequences for each class of genomic sequence (IGRs, TEs, and so forth) was calculated by Lu et al. and is shown in Fig. 1. The sRNA library from flower tissues contained over twice as many unique sRNAs as the one derived from 14-day-old seedlings, and much of that additional complexity came from IGRs. Lu et al. offer two possible explanations for this observation. The first is that TEs might be more strongly silenced in plant germline tissues. Consistent with this possibility, retrotransposons matched disproportionally more sRNAs in flowers than in seedlings (Fig. 1A). So perhaps the IGRs contain numerous cryptic retroelements that are more strongly silenced in flowers. The second proposed explanation for floral sRNA enrichment is that inflorescences contain a greater diversity of cell types than seedlings. Presumably these additional cell types express a wider variety of genes that could generate or be affected by sRNAs (7). Consistent with this data, 1.4-fold more genes are expressed in flowers than in seedlings, and some of these genes could be directly generating sRNA signatures (8). However, the IGRs are fourfold enriched in sRNA in flowers. What could be the source of these preferentially S PECIAL S ECTION A Genome sRNA Inflorescence sRNA Seedling B 4000 Sparse Moderate Dense 3000 Gene Pseudogene Intergenic Retroelement Transposon 2000 1000 Fig. 1. The distribution of distinct sRNA matches in the Arabidopsis genome. (A) Pie charts represent the distribution of distinct sRNA sequence signatures matching different components of the genome, rather than the abundance of each class of sRNA. The pie charts are scaled to indicate the greater number of distinct signatures in inflorescence relative to seedling RNA. (B) Distinct signature sequences for each annotated class are grouped according to their density: Sparse clusters have only a few sig- 0 Gene Pseudogene Intergenic Retroelement Transposon natures (though these can be abundant in the case of intergenic miRNA), whereas dense clusters have many distinct signatures per kilobase and are enriched in repeats as compared to genes and intergenic regions. www.sciencemag.org SCIENCE VOL 309 2 SEPTEMBER 2005 1525
RNA S PECIAL S ECTION expressed sequences? We know from wholegenome transcription tiling arrays that IGRs are rich with undescribed transcriptional units (9). These transcripts probably represent a mixture of unannotated protein coding genes and pseudogenes, members of unknown or sequencedivergent transposon families, transcribed repeats, and miRNAs. The physical distribution of sRNAs reveals more about the composition of the IGRs. Lu et al. classify sRNAs as belonging to sparse, moderate, and dense clusters in the genome, where sparse clusters have 1 to 10 distinct sRNA matches within 500 base pairs (bp) and probably represent miRNA homology. Moderate clusters have 11 to 20 matches per 500 bp, whereas dense clusters contain Q20 distinct sRNA matches. Centromeric repeats and TEs are enriched relative to genes in moderate and dense clusters. In contrast, IGRs are only slightly enriched in moderate and dense clusters, suggesting that centromeric repeats and TEs are not the predominant source of sRNAs in these domains (Fig. 1B). However, there are several hundred moderate and dense clusters in the IGRs. Because of their potential to prime iterative rounds of RdRP-mediated siRNA production and their robust association with previously cloned sRNA sequences (10), complex tandem repeats, which make up around 15% of the intergenic portion of the genome, merit special consideration as a potential source for these clusters. Lu et al. report a good correlation between tandem repeats and the presence of sRNAs. Tandem repeats have been implicated in several epigenetic phenomena, including imprinting and paramutation (11, 12), and sRNAs derived from tandem repeats could be involved in regulating germline-specific genes. Currently, 118 miRNAs are described in Arabidopsis that may regulate on the order of 700 genes (13). However, many genes that are not known or predicted targets of these miRNAs are up-regulated in RNAi-defective mutants (14), suggesting that additional miRNAs remain undiscovered. The sparse cluster component of IGRs reported by Lu et al. maycontainmanysuchmiRNAs.To investigate this possibility, a set of computational filters was used to capture most known miRNAs from the MPSS data, as well as potentially undescribed miRNAs, and these sequences were used to probe Northern blots of seedling and floral RNA from the rdr2 mutant, which is defective for an RNAdependent RNA polymerase that is responsible for producing most heterochromatic siRNAs. For several candidates, the sRNAs were found to be independent of RDR2, suggesting that they were previously undescribed miRNAs. Some of the candidate miRNAs were not found in the rice genome, suggesting that only the most deeply conserved miRNAs have thus far been identified in Arabidopsis. In plants, miRNAs act similarly to siRNAs in that they guide the cleavage of gene transcripts (15). Recently, a class of transitive miRNA-mediated regulation has been described. In these cases, miRNA-facilitated cleavage directs the recruitment of an RNAdependent RNA polymerase to generate dsRNAs, which are in turn processed into siRNAs. These siRNAs are able to transitively regulate other matching transcripts by recognizing sequences outside the original miRNA binding site and are thus called trans-acting siRNAs (or ta-siRNAs) (14). In all characterized instances of transitive RNAi, the primary transcript recognized by the miRNA is a noncoding gene, but in principle this mechanism could occur for any miRNA target. Lu et al. examined this possibility by looking for evidence of siRNAs generated from 61 known or predicted miRNA target genes. Except for PPR repeat genes (which have tandem repeats in their coding sequences), most miRNA targets exhibited little or no evidence of siRNA production, indicating that transitive RNAi occurs only in special instances, if at all. However, many ta-siRNA precursors were not annotated as transcriptional units in the Arabidopsis genome before they were discovered, and more may lie undiscoveredintheMPSSdata. Finally, nearly one in two annotated pseudogenes matched sRNAs, even when known TEs (an abundant source of pseudogenes) were excluded from the analysis. Only one in six genes matched sRNAs, suggesting that pseudogenes may be targeted specifically. What might the mechanism be? Expressed pseudogenes containing premature stop codons are expected to trigger nonsense-mediated decay (NMD) (16). However, the possibility that pseudogene sRNA sequences were RNA degradation products was excluded, because sRNA sequences derived from highly expressed genes with high turnover rates were not overrepresented. Instead, RNAs that experience premature termination may become substrates for RNAi through NMD. In most species, exonucleases are responsible for degrading mRNAs as part of the normal turnover process (16). In Drosophila, however, an unidentified endoribonuclease cleaves the nonsense transcript near the site of the premature termination codon (17). This may initiate RNAi-mediated degradation because the 5 and 3 ends of the cleavage products will be missing a polyA þ tail and 5 cap, respectively, resembling aberrant transcripts which are thought to be targeted by RNAi (1). Regardless of mechanism, if the 20- to 24- nucleotide RNAs generated from Arabidopsis pseudogenes are bona fide sRNAs, they could act transitively on transcripts from paralogous protein-coding genes by promoting cleavage or interfering with translation. More than half of the pseudogene sRNAs matched sequences elsewhere in the genome, indicating that this may be the case and suggesting a mechanism for coordinated trans-acting regulation of closely related members of gene families. This may also provide a mechanism for the origin of ta-siRNA, whose noncoding precursor RNA may be derived from ancient pseudogenes that are no longer recognizable except for the sRNA homology (14). What about sRNAs from genes? Small RNAs corresponding to heterochromatic transposons and repeats have been associated with chromatin modifications in the fission yeast Schizosaccharomyces pombe and with DNA methylation in plants, and the abundance of sRNAs from transposons and repeats supports a role in heterochromatic silencing (18). A significant proportion of genes in Arabidopsis also have some DNA methylation, although it is relatively sparse and is clustered near the 3 end of the genes (19, 20). However, our preliminary analysis indicates that partially methylated genes reported in microarray studies are not enriched in sRNAs, so that the role of sRNA from genes remains a mystery. Exceptions include PAI1 and PAI2, which have dense clusters of siRNA and are subject to RNA-dependent DNA methylation (3, 21). MPSS and other high-throughput sequencing technologies have the power to reveal the prevalence of sRNAs from essentially every class of sequence in the genome. Given that components of the RNAi machinery are found in almost all eukaryotes and many archaebacteria, the possibility of an early sRNA world is one that will receive attention in the future. References 1. D. Baulcombe, Trends Biochem. Sci. 30, 290 (2005). 2. P. D. Zamore, B. Haley, Science 309, 1519 (2005). 3. C. Lu et al., Science 309, 1567 (2005). 4. X. J. Wang, J. L. Reyes, N. H. Chua, T. Gaasterland, Genome Biol. 5, R65 (2004). 5. Z. Lippman, R. Martienssen, Nature 431, 364 (2004). 6. A. M. Gustafson et al., Nucleic Acids Res. 33, D637 (2005). 7. F. Meins Jr., A. Si-Ammour, T. Blevins, Annu. Rev. Cell Dev. Biol. 10.1146/annurev.cellbio.21.122303.114706. 8. B. C. Meyers et al., Genome Res. 14, 1641 (2004). 9. K. Yamada et al., Science 302, 842 (2003). 10. R. Martienssen, Z. Lippman, B. May, M. Ronemus, M. Vaughn, Cold Spring Harbor Symp. Quant. Biol. LXIX, 371 (2004). 11. T. Kinoshita et al., Science 303, 521 (2004). 12. M. Stam et al., Genetics 162, 917 (2002). 13. M. W. Jones-Rhoades, D. P. Bartel, Mol. Cell 14, 787 (2004). 14. E. Allen, Z. Xie, A. M. Gustafson, J. C. Carrington, Cell 121, 207 (2005). 15. G. Tang, P. D. Zamore, Methods Mol. Biol. 257, 223 (2004). 16. C. R. Alonso, Bioessays 27, 463 (2005). 17. M. A. Valencia-Sanchez, L. E. Maquat, Trends Cell Biol. 14, 594 (2004). 18. M. A. Matzke, J. A. Birchler, Nat. Rev. Genet. 6, 24 (2005). 19. Z. Lippman et al., Nature 430, 471 (2004). 20. R. K. Tran et al., Curr. Biol. 15, 154 (2005). 21. O. Mathieu, J. Bender, J. Cell Sci. 117, 4881 (2004). 10.1126/science.1117805 1526 2 SEPTEMBER 2005 VOL 309 SCIENCE www.sciencemag.org
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