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      Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana

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      1 , 2 , 1 , 3 , 4 ,
      Genome Biology
      BioMed Central

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          Abstract

          A new computational method for predicting cis-encoded natural antisense transcripts (NATs) in Arabidopsis identified 1,340 potential NAT pairs. The expression of both sense and antisense transcripts of 957 NAT pairs was confirmed, and analysis of MPSS data suggested that for most pairs one of the two transcripts is predominantly expressed in a tissue-specific manner.

          Abstract

          Background

          Natural antisense transcripts (NAT) are a class of endogenous coding or non-protein-coding RNAs with sequence complementarity to other transcripts. Several lines of evidence have shown that cis- and trans-NATs may participate in a broad range of gene regulatory events. Genome-wide identification of cis-NATs in human, mouse and rice has revealed their widespread occurrence in eukaryotes. However, little is known about cis-NATs in the model plant Arabidopsis thaliana.

          Results

          We developed a new computational method to predict and identify cis-encoded NATs in Arabidopsis and found 1,340 potential NAT pairs. The expression of both sense and antisense transcripts of 957 NAT pairs was confirmed using Arabidopsis full-length cDNAs and public massively parallel signature sequencing (MPSS) data. Three known or putative Arabidopsis imprinted genes have cis-antisense transcripts. Sequences and the genomic arrangement of two Arabidopsis NAT pairs are conserved in rice.

          Conclusion

          We combined information from full-length cDNAs and Arabidopsis genome annotation in our NAT prediction work and reported cis-NAT pairs that could not otherwise be identified by using one of the two datasets only. Analysis of MPSS data suggested that for most Arabidopsis cis-NAT pairs, there is predominant expression of one of the two transcripts in a tissue-specific manner.

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          Most cited references34

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          Non-coding RNA genes and the modern RNA world.

          S. Eddy (2001)
          Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins. However, almost all means of gene identification assume that genes encode proteins, so even in the era of complete genome sequences, ncRNA genes have been effectively invisible. Recently, several different systematic screens have identified a surprisingly large number of new ncRNA genes. Non-coding RNAs seem to be particularly abundant in roles that require highly specific nucleic acid recognition without complex catalysis, such as in directing post-transcriptional regulation of gene expression or in guiding RNA modifications.
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            Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

            We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.
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              The non-coding Air RNA is required for silencing autosomal imprinted genes.

              In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases, which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is correlated with repression of all three genes on the paternal allele; however, Air RNA overlaps just one of these genes in an antisense orientation. Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting.
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                Author and article information

                Journal
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1465-6906
                1465-6914
                2005
                15 March 2005
                : 6
                : 4
                : R30
                Affiliations
                [1 ]Laboratory of Computational Genomics, The Rockefeller University, New York, NY 10021, USA
                [2 ]Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
                [3 ]Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093, USA
                [4 ]Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10021, USA
                Article
                gb-2005-6-4-r30
                10.1186/gb-2005-6-4-r30
                1088958
                15833117
                dd462e48-6423-4dce-a267-de859c64ca21
                Copyright © 2005 Wang et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 December 2004
                : 7 February 2005
                : 25 February 2005
                Categories
                Research

                Genetics
                Genetics

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