title: Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell.
abstract: BACKGROUND
  RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.
  
  
  METHODS
  The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A-RNA interaction and RNA helicase A-stimulated viral RNA processes.
  
  
  RESULTS
  Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA(Lys3) annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A-HIV-1 RNA interaction in the cells.
  
  
  CONCLUSIONS
  The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.
  
  
  CONCLUSIONS
  The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.
authors.affiliation: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada. Electronic address: xingli107@gmail.com.
authors.forename: Li
authors.initials: L
authors.lastname: Xing
authors.affiliation: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Niu
authors.affiliation: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada. Electronic address: lawrence.kleiman@mcgill.ca.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Kleiman
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008239:Lysine; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
chemical_list: D009363:Neoplasm Proteins; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases; D008239:Lysine; D000409:Alanine
keywords: Double-stranded RNA binding domain; HIV-1; Helicase activity; Lysine; RNA helicase A