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      Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species

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          Abstract

          Background

          Recent studies in budding yeast have shown that antisense transcription occurs at many loci. However, the functional role of antisense transcripts has been demonstrated only in a few cases and it has been suggested that most antisense transcripts may result from promiscuous bi-directional transcription in a dense genome.

          Results

          Here, we use strand-specific RNA sequencing to study anti-sense transcription in Saccharomyces cerevisiae. We detect 1,103 putative antisense transcripts expressed in mid-log phase growth, ranging from 39 short transcripts covering only the 3' UTR of sense genes to 145 long transcripts covering the entire sense open reading frame. Many of these antisense transcripts overlap sense genes that are repressed in mid-log phase and are important in stationary phase, stress response, or meiosis. We validate the differential regulation of 67 antisense transcripts and their sense targets in relevant conditions, including nutrient limitation and environmental stresses. Moreover, we show that several antisense transcripts and, in some cases, their differential expression have been conserved across five species of yeast spanning 150 million years of evolution. Divergence in the regulation of antisense transcripts to two respiratory genes coincides with the evolution of respiro-fermentation.

          Conclusions

          Our work provides support for a global and conserved role for antisense transcription in yeast gene regulation.

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          Most cited references26

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          The transcriptional landscape of the yeast genome defined by RNA sequencing.

          The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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            Direct multiplexed measurement of gene expression with color-coded probe pairs.

            We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.
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              Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

              Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright 1999 John Wiley & Sons, Ltd.
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                Author and article information

                Journal
                Genome Biol
                Genome Biology
                BioMed Central
                1465-6906
                1465-6914
                2010
                26 August 2010
                : 11
                : 8
                : R87
                Affiliations
                [1 ]Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
                [2 ]Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, 68-132, Cambridge, MA 02139, USA
                [3 ]School of Engineering and Computer Science, Hebrew University, Ross Building, Givat Ram Campus, Jerusalem, 91904, Israel
                [4 ]Alexander Silberman Institute of Life Sciences, Hebrew University, Edmond J Safra Campus, Givat Ram, Jerusalem, 91904, Israel
                Article
                gb-2010-11-8-r87
                10.1186/gb-2010-11-8-r87
                2945789
                20796282
                0da127e8-c27d-44d0-8e6f-eae3b4356916
                Copyright ©2010 Yassour 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
                : 15 February 2010
                : 26 July 2010
                : 26 August 2010
                Categories
                Research

                Genetics
                Genetics

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