Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs.
How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Psi residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Psi residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Psi residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3'-guide sequence is more important than that formed with the 5'-guide sequence. Surprisingly, we showed that a sRNA-rRNA interaction with the targeted uridine in a single-stranded 5'-UNN-3' trinucleotide instead of the canonical 5'-UN-3' dinucleotide is functional.
Muller
Sébastien
S
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Nancy Université, Faculté des Sciences et Techniques, 54506 Vandoeuvre-lès-Nancy, France.
Leclerc
Fabrice
F
Behm-Ansmant
Isabelle
I
Fourmann
Jean-Baptiste
JB
Charpentier
Bruno
B
Branlant
Christiane
C
D001483:Base Sequence; D019295:Computational Biology; D017124:Conserved Sequence; D023281:Genomics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011560:Pseudouridine / Q000032:analysis*; D019711:Pyrococcus / Q000235:genetics; D043903:Pyrococcus abyssi / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D012984:Software
D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D020537:RNA, Small Nucleolar; D011560:Pseudouridine