[Show abstract][Hide abstract] ABSTRACT: Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently approximately 240 microRNAs (miRNAs) have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, we found a unique class of miRNAs that derive from natural cis-antisense transcript pairs. This configuration generates miRNAs that can perfectly match their targets. We provide evidence that the miRNAs function by inducing mRNA cleavage in the middle of their complementary site. Their production requires Dicer-like 1 (DCL1) activity, which is essential for canonical miRNA biogenesis. All of the natural antisense miRNAs (nat-miRNAs) identified in this study have large introns in their precursors that appear critical for nat-miRNA evolution and for the formation of functional miRNA loci. These findings suggest that other natural cis-antisense loci with similar exon-intron arrangements could be another source of miRNA genes.
Full-text · Article · Apr 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Identification of all expressed transcripts in a sequenced genome is essential both for genome analysis and for realization of the goals of systems biology. We used the transcriptional profiling technology called 'massively parallel signature sequencing' to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). We sequenced 46,971,553 mRNA transcripts from 22 libraries, and 2,953,855 small RNAs from 3 libraries. The data demonstrate widespread transcription throughout the genome, including sense expression of at least 25,500 annotated genes and antisense expression of nearly 9,000 annotated genes. An additional set of approximately 15,000 mRNA signatures mapped to unannotated genomic regions. The majority of the small RNA data represented lower abundance short interfering RNAs that match repetitive sequences, intergenic regions and genes. Among these, numerous clusters of highly regulated small RNAs were readily observed. We developed a genome browser (http://mpss.udel.edu/rice) for public access to the transcriptional profiling data for this important crop.
Full-text · Article · May 2007 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.