title: Intermolecular interactions within the abundant DEAD-box protein Dhh1 regulate its activity in vivo.
abstract: Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.
authors.affiliation: Center for Eukaryotic Gene Regulation, Penn State University, University Park, Pennsylvania 16802, USA.
authors.forename: Arnob
authors.initials: A
authors.lastname: Dutta
authors.forename: Suting
authors.initials: S
authors.lastname: Zheng
authors.forename: Deepti
authors.initials: D
authors.lastname: Jain
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Reese
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D053487:DEAD-box RNA Helicases