title: Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop.
abstract: In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNA(Thr) species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNA(Thr) species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site.
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering and Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hirata
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Kitajima
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
mesh_terms: D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020745:Genome, Archaeal; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D012363:RNA, Transfer, Thr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D012322:RNA Precursors; D012363:RNA, Transfer, Thr; D004722:Endoribonucleases; C051012:splicing endonuclease