title: Pseudouridylation of U(35) in the anticodon of Arabidopsis thaliana pre-tRNA(Tyr) depends on length rather than structure of an intron.
abstract: In order to establish the structure and sequence requirements for pseudouridine (Psi(35)) biosynthesis in Arabidopsis thaliana tRNA(Tyr) five mutants of nuclear pre-tRNA(Tyr) have been prepared and analyzed: DeltaI-tRNA(Tyr) transcript depleted of an intron, and 5UI, 7UI, 9UI and 12UI transcripts containing tracts of five, seven, nine and 12 U residues, respectively, instead of the wild type tRNA(Tyr) intron. The in vitro transcripts were incubated in a lupin seed extract containing Psi(35) synthase activity, and those containing an artificial intron composed of 12 or nine U residues turned out to be good substrates for Psi(35) synthase. The transcript with an intron composed of seven uridine residues was pseudouridylated up to 40%, whereas the remaining two were not pseudouridylated at all. The secondary structures of all transcripts were determined using enzymatic and chemical probes: S(1), V(1), T(1), A, P(1) and Pb(2+). All mutant pre-tRNAs show similar structural features: their anticodon arm contains a five base pair stem and a large loop which consists of five natural tRNA(Tyr) AC loop nucleotides to which five, seven, nine and 12 U residues are added. As the structure of the wild type pre-tRNA(Tyr) is different we propose that the role of its intron in the process of U(35) pseudouridylation is simply to expand the anticodon region to the required critical length.
authors.affiliation: Department of Gene Expression, Adam Mickiewicz University, Poznań, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Pieńkowska
authors.forename: Daniel
authors.initials: D
authors.lastname: Michałowski
authors.forename: Wlodzimierz J
authors.initials: WJ
authors.lastname: Krzyzosiak
authors.forename: Zofia
authors.initials: Z
authors.lastname: Szweykowska-Kulińska
mesh_terms: D000926:Anticodon; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D011560:Pseudouridine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D012322:RNA Precursors; D012365:RNA, Transfer, Tyr; C030449:pre-tRNA(Tyr); D011560:Pseudouridine; D019751:Intramolecular Transferases; C096995:pseudouridine synthases