Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin. Issue 6 (27th March 2016)

• Record Type: Journal Article
• Authors: Ma, Wai Kit; Paudel, Bishnu P.; Xing, Zheng; Sabath, Ivan G.; Rueda, David; Tran, Elizabeth J.
• Is Part Of: Journal of molecular biology. Volume 428:Issue 6(2016:Mar. 27)
• Page Start: 1091
• Page End: 1106
• Publication Date: 2016-03-27
• Subjects: RNP RNA–protein -- smFRET single-molecule fluorescence resonance energy transfer -- ChIP chromatin immunoprecipitation -- RIP RNA immunoprecipitation -- BSA bovine serum albumin; DEAD-box -- helicase -- RNA–protein complex -- chromatin -- RNA; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.02.005
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Highlights: Dbp2 associates with chromatin in an RNA-dependent manner. Loss of Yra1–Dbp2 interaction leads to over-accumulation of Dbp2 on mRNA. Yra1 inhibits duplex unwinding by reducing RNA-binding activity of Dbp2. Yra1-dependent inhibition of Dbp2 prevents aberrant mRNA stabilization. Regulation of Dbp2 is critical for maintaining proper gene expression.