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      High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes

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          Abstract

          In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.

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          Prediction of mammalian microRNA targets.

          MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.
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            Vienna RNA secondary structure server.

            The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a set of aligned sequences and the design of sequences that will fold into a predefined structure. All three services can be accessed via the Vienna RNA web server at http://rna.tbi.univie.ac.at/.
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              Mechanisms of gene silencing by double-stranded RNA.

              Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS ONE
                plos
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2007
                14 February 2007
                : 2
                : 2
                : e219
                Affiliations
                [1 ]Center for Genome Research and Biocomputing, Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America
                [2 ]Molecular and Cellular Biology Graduate Program, Oregon State University, Corvallis, Oregon, United States of America
                [3 ]Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America
                Michigan State University, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: carrington@ 123456cgrb.oregonstate.edu

                Conceived and designed the experiments: JC JD SG KK NF EC MH. Performed the experiments: SG NF EC MH TL. Analyzed the data: JC KK NF CS JC SG MH. Wrote the paper: JC KK NF.

                Article
                06-PONE-RA-00472R1
                10.1371/journal.pone.0000219
                1790633
                17299599
                024dd3a9-8ef8-410d-9ca5-078a376cf386
                Fahlgren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 11 December 2006
                : 24 January 2007
                Page count
                Pages: 14
                Categories
                Research Article
                Computational Biology/Genomics
                Evolutionary Biology/Plant Genomes and Evolution
                Genetics and Genomics/Functional Genomics
                Plant Biology/Plant Genomes and Evolution
                Plant Biology/Plant-Biotic Interactions

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