Pseudouridylation of U35 in the anticodon of Arabidopsis thaliana pre-tRNATyr depends on length rather than structure of an intron

Abstract

<p class="first" id="d3548410e73">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. </p>