Functional characterization of XendoU, the endoribonuclease involved in small nucleolar RNA biosynthesis.
XendoU is the endoribonuclease involved in the biosynthesis of a specific subclass of Xenopus laevis intron-encoded small nucleolar RNAs. XendoU has no homology to any known cellular RNase, although it has sequence similarity with proteins tentatively annotated as serine proteases. It has been recently shown that XendoU represents the cellular counterpart of a nidovirus replicative endoribonuclease (NendoU), which plays a critical role in viral replication and transcription. In this paper, we combined prediction and experimental data to define the amino acid residues directly involved in XendoU catalysis. Specifically, we find that XendoU residues Glu-161, Glu-167, His-162, His-178, and Lys-224 are essential for RNA cleavage, which occurs in the presence of manganese ions. Furthermore, we identified the RNA sequence required for XendoU binding and showed that the formation of XendoU-RNA complex is Mn2+-independent.
Gioia
Ubaldo
U
Institute Pasteur-Fondazione Cenci Bolognetti, Department of Genetics and Molecular Biology, University La Sapienza, p.le A. Moro 5, 00185 Rome, Italy.
Laneve
Pietro
P
Dlakic
Mensur
M
Arceci
Massimo
M
Bozzoni
Irene
I
Caffarelli
Elisa
E
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry / Q000502:physiology*; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007438:Introns; D008239:Lysine / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012342:RNA, Small Nuclear / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000502:physiology*; D014982:Xenopus laevis
D017931:DNA Primers; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D018698:Glutamic Acid; D008345:Manganese; D006639:Histidine; D004722:Endoribonucleases; C475059:ENDOU protein, Xenopus; D008239:Lysine