title: Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.
abstract: RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071001, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO, 63110, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Burclaff
authors.affiliation: Department of Biological Sciences, Marquette University, Milwaukee, WI, 53233, United States of America.
authors.forename: James T
authors.initials: JT
authors.lastname: Anderson
mesh_terms: D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D020871:RNA Stability; D012331:RNA, Fungal / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D012331:RNA, Fungal; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
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