title: Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.
abstract: RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2.
authors.affiliation: Department of Biochemistry, Purdue University, BCHM 305, 175 S. University Street, West Lafayette, IN 47907-2063, USA.
authors.forename: Wai Kit
authors.initials: WK
authors.lastname: Ma
authors.affiliation: Department of Medicine, Section of Virology, and Single Molecule Imaging Group, MRC-Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.
authors.forename: Bishnu P
authors.initials: BP
authors.lastname: Paudel
authors.affiliation: Department of Biochemistry, Purdue University, BCHM 305, 175 S. University Street, West Lafayette, IN 47907-2063, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Xing
authors.affiliation: Department of Biochemistry, Purdue University, BCHM 305, 175 S. University Street, West Lafayette, IN 47907-2063, USA.
authors.forename: Ivan G
authors.initials: IG
authors.lastname: Sabath
authors.affiliation: Department of Medicine, Section of Virology, and Single Molecule Imaging Group, MRC-Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address: david.rueda@imperial.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Rueda
authors.affiliation: Department of Biochemistry, Purdue University, BCHM 305, 175 S. University Street, West Lafayette, IN 47907-2063, USA; Purdue University Center for Cancer Research, Purdue University, Hansen Life Sciences Research Building, Room 141, 201 S. University Street, West Lafayette, IN 47907-2064, USA. Electronic address: ejtran@purdue.edu.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Tran
mesh_terms: D002843:Chromatin / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; C105887:YRA1 protein, S cerevisiae; C026452:messenger ribonucleoprotein; C438898:Dbp2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: DEAD-box; RNA; RNA–protein complex; chromatin; helicase