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Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs.

Muller S, Leclerc F, Behm-Ansmant I, Fourmann JB, Charpentier B, Branlant C - Nucleic Acids Res. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Nancy Université, Faculté des Sciences et Techniques, 54506 Vandoeuvre-lès-Nancy, France.

ABSTRACT
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.

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Sequences and proposed secondary structures of the seven pyrococcal H/ACA sRNA candidates. The sequences and proposed secondary structures for the seven candidate P. abyssi sRNAs, Pab21 (A), Pab35 (B), Pab40 (C), Pab91 (D), Pab105 (E), Pab160 (F) and Pab19 (G) sRNAs, are shown. The ANA sequence at the 3′ end of the RNA and the K-turn or K-loop motif in the apical part of the H/ACA motif are boxed. Base substitutions in the P. furiosus (P.f.), P. horikoshii (P.h.) and T. kodakarensis (T.k.) (25) sRNAs are shown. The two putative foldings, which can be proposed for sRNA Pab21 and for motif 2 in sRNA Pab40, are shown (insets in panels A and C). Only the conformation shown in the entire molecule turned to be functional.
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Figure 2: Sequences and proposed secondary structures of the seven pyrococcal H/ACA sRNA candidates. The sequences and proposed secondary structures for the seven candidate P. abyssi sRNAs, Pab21 (A), Pab35 (B), Pab40 (C), Pab91 (D), Pab105 (E), Pab160 (F) and Pab19 (G) sRNAs, are shown. The ANA sequence at the 3′ end of the RNA and the K-turn or K-loop motif in the apical part of the H/ACA motif are boxed. Base substitutions in the P. furiosus (P.f.), P. horikoshii (P.h.) and T. kodakarensis (T.k.) (25) sRNAs are shown. The two putative foldings, which can be proposed for sRNA Pab21 and for motif 2 in sRNA Pab40, are shown (insets in panels A and C). Only the conformation shown in the entire molecule turned to be functional.

Mentions: In order to make a link between H/ACA sRNAs and pseudouridylation in rRNAs, in the P. abyssi species, we first made a complete identification of H/ACA–sRNA motifs in P. abyssi. This was done in two steps because, when we started this study, only a limited number of archaeal H/ACA sRNAs had been identified. As a first step, we applied a phylogenetic approach to the Pyrococcus genus. The idea was that DNA segments, which link ORFs and/or template sequences for stable known RNAs (rRNAs, tRNAs, RNase P, 7S RNA) (Inter-Coding-Regions, ICRs) and that bear long stretches of conserved sequences in three Pyrococcus species P. abyssi, P. furiosus and P. horikoshii, may correspond to genes for functional non-coding RNAs. Based on the few H/ACA sRNAs known at that time (three from A. fulgidus) (21) some characteristic features of H/ACA sRNA motifs could be delineated. They were used to build an RNAMOT descriptor for the search of H/ACA sRNA genes. Then, RNAMOT profiles were built for the search of the possible rRNA target sites of each candidate H/ACA motif. By using this approach (see the details in Materials and Methods), we detected five putative H/ACA sRNA genes, that were common to the three species. Four of them have been recently characterized in P. furiosus by the use of a computational approach based on G/C content analysis, namely, the Pf1, Pf3, Pf6 and Pf7 sRNAs (22,49). The counterparts that we identified in P. abyssi and P. horikoshii are designated as Pab21, Pab105, Pab35 and Pab40 sRNAs (P. abyssi) and Pho21, Pho 105, Pho35 and Pho40 sRNAs (P. horikoshii), respectively (Figure 2). The fifth common H/ACA sRNA, which had not been characterized by other teams, is denoted Pab91, Pfu91 and Pho91 in P. abyssi, P. furiosus and P. horikoshii, respectively. We previously used it to settle conditions for in vitro reconstitution of active H/ACA sRNPs (23). Taking into account the numerous compensatory base-pair mutations in the three species studied and in T. kodakarensis, we could propose relevant secondary structures for each of the five sRNAs (Figure 2). Only for sRNA Pab21 and for motif 2 in sRNA Pab40, it was difficult to make a choice between two possible 2D structures that were both containing a K-loop motif (Figure 2). Production of the H/ACA sRNAs Pab21, Pab35, Pab40, Pab91 and Pab105 in P. abyssi was verified by Northern blot analysis (Figure 3), and two forms of Pab21 sRNA with or without the C/D box motif were found to be present in vivo (Figure 3).Figure 2.


Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs.

Muller S, Leclerc F, Behm-Ansmant I, Fourmann JB, Charpentier B, Branlant C - Nucleic Acids Res. (2008)

Sequences and proposed secondary structures of the seven pyrococcal H/ACA sRNA candidates. The sequences and proposed secondary structures for the seven candidate P. abyssi sRNAs, Pab21 (A), Pab35 (B), Pab40 (C), Pab91 (D), Pab105 (E), Pab160 (F) and Pab19 (G) sRNAs, are shown. The ANA sequence at the 3′ end of the RNA and the K-turn or K-loop motif in the apical part of the H/ACA motif are boxed. Base substitutions in the P. furiosus (P.f.), P. horikoshii (P.h.) and T. kodakarensis (T.k.) (25) sRNAs are shown. The two putative foldings, which can be proposed for sRNA Pab21 and for motif 2 in sRNA Pab40, are shown (insets in panels A and C). Only the conformation shown in the entire molecule turned to be functional.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Sequences and proposed secondary structures of the seven pyrococcal H/ACA sRNA candidates. The sequences and proposed secondary structures for the seven candidate P. abyssi sRNAs, Pab21 (A), Pab35 (B), Pab40 (C), Pab91 (D), Pab105 (E), Pab160 (F) and Pab19 (G) sRNAs, are shown. The ANA sequence at the 3′ end of the RNA and the K-turn or K-loop motif in the apical part of the H/ACA motif are boxed. Base substitutions in the P. furiosus (P.f.), P. horikoshii (P.h.) and T. kodakarensis (T.k.) (25) sRNAs are shown. The two putative foldings, which can be proposed for sRNA Pab21 and for motif 2 in sRNA Pab40, are shown (insets in panels A and C). Only the conformation shown in the entire molecule turned to be functional.
Mentions: In order to make a link between H/ACA sRNAs and pseudouridylation in rRNAs, in the P. abyssi species, we first made a complete identification of H/ACA–sRNA motifs in P. abyssi. This was done in two steps because, when we started this study, only a limited number of archaeal H/ACA sRNAs had been identified. As a first step, we applied a phylogenetic approach to the Pyrococcus genus. The idea was that DNA segments, which link ORFs and/or template sequences for stable known RNAs (rRNAs, tRNAs, RNase P, 7S RNA) (Inter-Coding-Regions, ICRs) and that bear long stretches of conserved sequences in three Pyrococcus species P. abyssi, P. furiosus and P. horikoshii, may correspond to genes for functional non-coding RNAs. Based on the few H/ACA sRNAs known at that time (three from A. fulgidus) (21) some characteristic features of H/ACA sRNA motifs could be delineated. They were used to build an RNAMOT descriptor for the search of H/ACA sRNA genes. Then, RNAMOT profiles were built for the search of the possible rRNA target sites of each candidate H/ACA motif. By using this approach (see the details in Materials and Methods), we detected five putative H/ACA sRNA genes, that were common to the three species. Four of them have been recently characterized in P. furiosus by the use of a computational approach based on G/C content analysis, namely, the Pf1, Pf3, Pf6 and Pf7 sRNAs (22,49). The counterparts that we identified in P. abyssi and P. horikoshii are designated as Pab21, Pab105, Pab35 and Pab40 sRNAs (P. abyssi) and Pho21, Pho 105, Pho35 and Pho40 sRNAs (P. horikoshii), respectively (Figure 2). The fifth common H/ACA sRNA, which had not been characterized by other teams, is denoted Pab91, Pfu91 and Pho91 in P. abyssi, P. furiosus and P. horikoshii, respectively. We previously used it to settle conditions for in vitro reconstitution of active H/ACA sRNPs (23). Taking into account the numerous compensatory base-pair mutations in the three species studied and in T. kodakarensis, we could propose relevant secondary structures for each of the five sRNAs (Figure 2). Only for sRNA Pab21 and for motif 2 in sRNA Pab40, it was difficult to make a choice between two possible 2D structures that were both containing a K-loop motif (Figure 2). Production of the H/ACA sRNAs Pab21, Pab35, Pab40, Pab91 and Pab105 in P. abyssi was verified by Northern blot analysis (Figure 3), and two forms of Pab21 sRNA with or without the C/D box motif were found to be present in vivo (Figure 3).Figure 2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Nancy Université, Faculté des Sciences et Techniques, 54506 Vandoeuvre-lès-Nancy, France.

ABSTRACT
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.

Show MeSH