Genome‐wide function of THO/TREX in active genes prevents R‐loop‐dependent replication obstacles

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription‐associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)‐chip studies reveal that the Hpr1 component of THO and the Sub2 RNA‐dependent ATPase have genome‐wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5′ → 3′ gradient. This is accompanied by a genome‐wide impact of THO–Sub2 deletions on expression of highly expressed, long and high G+C‐content genes. Importantly, ChIP‐chips reveal an over‐recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome‐wide function for THO–Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription‐mediated replication obstacles, including R‐loops.

[1]  R. Luna,et al.  Interdependence between transcription and mRNP processing and export, and its impact on genetic stability. , 2005, Molecular cell.

[2]  K. Kurokawa,et al.  Genome-Organizing Factors Top2 and Hmo1 Prevent Chromosome Fragility at Sites of S phase Transcription , 2009, Cell.

[3]  J. Manley,et al.  Inactivation of the SR Protein Splicing Factor ASF/SF2 Results in Genomic Instability , 2005, Cell.

[4]  Jin-Qiu Zhou,et al.  The Saccharomyces Pif1p DNA Helicase and the Highly Related Rrm3p Have Opposite Effects on Replication Fork Progression in Ribosomal DNA , 2000, Cell.

[5]  Andrés Aguilera,et al.  A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation‐associated recombination , 1998, The EMBO journal.

[6]  Marco Foiani,et al.  Maintaining genome stability at the replication fork , 2010, Nature Reviews Molecular Cell Biology.

[7]  A. Aguilera,et al.  Molecular evidence for a positive role of Spt4 in transcription elongation , 2003, The EMBO journal.

[8]  M. Grunstein,et al.  Mapping DNA Interaction Sites of Chromosomal Proteins , 1999 .

[9]  A. Jansen,et al.  Genes with internal repeats require the THO complex for transcription , 2006, Proceedings of the National Academy of Sciences.

[10]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[11]  Daniel Zenklusen,et al.  Stable mRNP Formation and Export Require Cotranscriptional Recruitment of the mRNA Export Factors Yra1p and Sub2p by Hpr1p , 2002, Molecular and Cellular Biology.

[12]  D. Libri,et al.  Multiple roles for the yeast SUB2/yUAP56 gene in splicing. , 2001, Genes & development.

[13]  A. Aguilera,et al.  AID Induces Double-Strand Breaks at Immunoglobulin Switch Regions and c-MYC Causing Chromosomal Translocations in Yeast THO Mutants , 2011, PLoS genetics.

[14]  Andrés Aguilera,et al.  The interface between transcription and mRNP export: from THO to THSC/TREX-2. , 2010, Biochimica et biophysica acta.

[15]  Lara K. Goudsouzian,et al.  The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes. , 2003, Molecular cell.

[16]  B. Gómez-González,et al.  The S-Phase Checkpoint Is Required To Respond to R-Loops Accumulated in THO Mutants , 2009, Molecular and Cellular Biology.

[17]  S. Lacadie,et al.  Biochemical analysis of TREX complex recruitment to intronless and intron‐containing yeast genes , 2004, The EMBO journal.

[18]  A. Aguilera.  mRNA processing and genomic instability , 2005, Nature Structural &Molecular Biology.

[19]  F. Prado,et al.  Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex , 2006, Molecular and Cellular Biology.

[20]  M. Lieber,et al.  R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells , 2003, Nature Immunology.

[21]  M. Lieber,et al.  Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism. , 2003, DNA repair.

[22]  Kevin Struhl,et al.  TREX is a conserved complex coupling transcription with messenger RNA export , 2002, Nature.

[23]  R. Luna,et al.  An hpr1 Point Mutation That Impairs Transcription and mRNP Biogenesis without Increasing Recombination , 2006, Molecular and Cellular Biology.

[24]  Stefan Kemmler,et al.  Sem1 is a functional component of the nuclear pore complex–associated messenger RNA export machinery , 2009, The Journal of cell biology.

[25]  R. Luna,et al.  Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity , 2008, Molecular Genetics and Genomics.

[26]  C. Li,et al.  Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Krogan,et al.  Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes , 2004, The EMBO journal.

[28]  Andrés Aguilera,et al.  Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. , 2003, Molecular cell.

[29]  F. Prado,et al.  Impairment of replication fork progression mediates RNA polII transcription‐associated recombination , 2005, The EMBO journal.

[30]  H. Aburatani,et al.  Genomic approach for the understanding of dynamic aspect of chromosome behavior. , 2006, Methods in enzymology.

[31]  B. Michel,et al.  The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo , 2009, The EMBO journal.

[32]  M. Grunstein,et al.  Mapping DNA interaction sites of chromosomal proteins. Crosslinking studies in yeast. , 1999, Methods in molecular biology.

[33]  A. Aguilera,et al.  Molecular Evidence That the Eukaryotic THO/TREX Complex Is Required for Efficient Transcription Elongation* , 2003, Journal of Biological Chemistry.

[34]  Amy C. Kelly,et al.  Saccharomyces cerevisiae , 2013, Prion.

[35]  P. Hanawalt,et al.  Mechanisms and implications of transcription blockage by guanine-rich DNA sequences , 2010, Proceedings of the National Academy of Sciences.

[36]  Anna Azvolinsky,et al.  The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes. , 2006, Genes & development.

[37]  Katsuhiko Shirahige,et al.  Relationship between G+C content, ORF‐length and mRNA concentration in Saccharomyces cerevisiae , 2003, Yeast.

[38]  A. Aguilera,et al.  Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex , 2007, Proceedings of the National Academy of Sciences.

[39]  Johannes Söding,et al.  Uniform transitions of the general RNA polymerase II transcription complex , 2010, Nature Structural &Molecular Biology.

[40]  Takashi Horiuchi,et al.  Transcription-dependent recombination and the role of fork collision in yeast rDNA. , 2003, Genes & development.

[41]  R. Wollman,et al.  A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. , 2009, Molecular cell.

[42]  R. Luna,et al.  The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability , 2002, The EMBO journal.

[43]  R. Xu,et al.  Crystal structure of the human ATP-dependent splicing and export factor UAP56. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Katsuhiko Shirahige,et al.  Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. , 2007, Genes & development.

[45]  Trevor Lithgow,et al.  A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae , 2000, The EMBO journal.

[46]  H. Klein,et al.  HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene , 1990, Molecular and cellular biology.

[47]  S. Chávez,et al.  Hpr1 Is Preferentially Required for Transcription of Either Long or G+C-Rich DNA Sequences in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[48]  P. Hanawalt,et al.  Anchoring nascent RNA to the DNA template could interfere with transcription. , 2011, Biophysical journal.

[49]  T. Köcher,et al.  Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster , 2004, Nature Structural &Molecular Biology.

[50]  Rosa Luna,et al.  Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus , 2008, Chromosoma.

[51]  M. Lieber,et al.  G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter , 2009, Molecular and Cellular Biology.

[52]  Danny Reinberg,et al.  Elongation by RNA polymerase II: the short and long of it. , 2004, Genes & development.

[53]  P. Silver,et al.  Messenger RNAs are recruited for nuclear export during transcription. , 2001, Genes & development.

[54]  A. Antoni,et al.  A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. , 2000, Gene.

[55]  Rosa Luna,et al.  The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability. , 2008, Molecular biology of the cell.

[56]  P. Giresi,et al.  Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae. , 2009, Molecular cell.

[57]  A. Aguilera,et al.  Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New insights into transcription-associated recombination. , 2003, Genetics.

[58]  S. Mirkin,et al.  Mechanisms of Transcription-Replication Collisions in Bacteria , 2005, Molecular and Cellular Biology.

[59]  K. Kitada,et al.  Mapping of early firing origins on a replication profile of budding yeast , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[60]  B. Gómez-González,et al.  Genome instability: a mechanistic view of its causes and consequences , 2008, Nature Reviews Genetics.

[61]  Katsuhiko Shirahige,et al.  S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex , 2003, Nature.

[62]  M. Lopes,et al.  Methods to study replication fork collapse in budding yeast. , 2006, Methods in enzymology.

[63]  A. Aguilera,et al.  Stimulation of direct-repeat recombination by RNA polymerase III transcription. , 2009, DNA repair.

[64]  Kevin Struhl,et al.  Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. , 2005, Molecular cell.

[65]  Frédéric Devaux,et al.  THO/Sub2p Functions to Coordinate 3′-End Processing with Gene-Nuclear Pore Association , 2008, Cell.