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Human Senataxin Resolves RNA/DNA Hybrids Formed at Transcriptional Pause Sites to Promote Xrn2-Dependent Termination

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      Summary

      We present a molecular dissection of pause site-dependent transcriptional termination for mammalian RNA polymerase II (Pol II)-transcribed genes. We show that nascent transcripts form RNA/DNA hybrid structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein associated with AOA2/ALS4 neurodegenerative disorders, acts to resolve these R-loop structures and by so doing allows access of the 5 –3 exonuclease Xrn2 at 3 cleavage poly(A) sites. This affords 3 transcript degradation and consequent Pol II termination. In effect, R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process.

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      Highlights

      ► Senataxin is required for pause-dependent Pol II termination in human genes ► R-loops accumulate over G-rich pause regions and are required for termination ► R-loops dependent on transcription, functional poly(A) signal, and pause elements ► Senataxin resolves R-loops to allow Xrn2 to mediate Pol II release from the gene

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      Most cited references 52

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      Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.

      RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
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        Pre-mRNA processing reaches back to transcription and ahead to translation.

        The pathway from gene activation in the nucleus to mRNA translation and decay at specific locations in the cytoplasm is both streamlined and highly interconnected. This review discusses how pre-mRNA processing, including 5' cap addition, splicing, and polyadenylation, contributes to both the efficiency and fidelity of gene expression. The connections of pre-mRNA processing to upstream events in transcription and downstream events, including translation and mRNA decay, are elaborate, extensive, and remarkably interwoven.
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          Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.

           Q. Li,  James Manley (2005)
          SR proteins constitute a family of pre-mRNA splicing factors now thought to play several roles in mRNA metabolism in metazoan cells. Here we provide evidence that a prototypical SR protein, ASF/SF2, is unexpectedly required for maintenance of genomic stability. We first show that in vivo depletion of ASF/SF2 results in a hypermutation phenotype likely due to DNA rearrangements, reflected in the rapid appearance of DNA double-strand breaks and high-molecular-weight DNA fragments. Analysis of DNA from ASF/SF2-depleted cells revealed that the nontemplate strand of a transcribed gene was single stranded due to formation of an RNA:DNA hybrid, R loop structure. Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes. Indicative of a direct role, ASF/SF2 prevented R loop formation in a reconstituted in vitro transcription reaction. Our results support a model by which recruitment of ASF/SF2 to nascent transcripts by RNA polymerase II prevents formation of mutagenic R loop structures.
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            Author and article information

            Affiliations
            [1 ]Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
            Author notes
            []Corresponding author nicholas.proudfoot@ 123456path.ox.ac.uk
            [∗∗ ]Corresponding author natalia.gromak@ 123456path.ox.ac.uk
            Contributors
            Journal
            Mol Cell
            Mol. Cell
            Molecular Cell
            Cell Press
            1097-2765
            1097-4164
            24 June 2011
            24 June 2011
            : 42
            : 6
            : 794-805
            3145960
            21700224
            MOLCEL3891
            10.1016/j.molcel.2011.04.026
            © 2011 ELL & Excerpta Medica.

            This document may be redistributed and reused, subject to certain conditions.

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            Molecular biology

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