title: Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease.
abstract: Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.
authors: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA. MNASHIMOTO@chemgate.byu.edu
authors: M
authors: M
authors: Nashimoto
authors: M
authors: M
authors: Tamura
authors: R L
authors: RL
authors: Kaspar
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015347:RNA Probes; D012347:RNA, Transfer, Arg / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013552:Swine
chemical_list: D012322:RNA Precursors; D015347:RNA Probes; D012347:RNA, Transfer, Arg; D004722:Endoribonucleases; C045782:3'-pre-tRNase