title: A homolog of lariat-debranching enzyme modulates turnover of branched RNA.
abstract: Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudo-metallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5'-exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Garrey
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6, Canada.
authors.forename: Adam
authors.initials: A
authors.lastname: Katolik
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Mantas
authors.initials: M
authors.lastname: Prekeris
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Xueni
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Kerri
authors.initials: K
authors.lastname: York
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Bernards
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6, Canada.
authors.forename: Masad J
authors.initials: MJ
authors.lastname: Damha
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Hesselberth
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D007438:Introns; D008969:Molecular Sequence Data; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000502:physiology*; D012331:RNA, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017381:Spliceosomes / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D012331:RNA, Fungal; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme
keywords: Dbr1; Nineteen Complex; branched RNA; splicing