Dataset Information

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

ABSTRACT: Pseudouridine (Psi) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Psi synthase bind directly the sRNA; aCBF5 also interacts directly and independently with aNOP10 and aGAR1. Presence of aCBF5, aNOP10 and a U residue at the pseudouridylation site in the target RNA are required for RNA target recruitment. In agreement, we found that the aCBF5-aNOP10 pair is the minimal set of proteins needed for the formation of a particle active for pseudouridylation. However, particles more efficient in targeted pseudouridylation can be formed with the addition of proteins L7Ae and/or aGAR1. Although necessary for optimal activity, the conserved ACA motif in the sRNA was found to be not essential.

SUBMITTER: Charpentier B

PROVIDER: S-EPMC1142404 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

Charpentier Bruno B Muller Sébastien S Branlant Christiane C

Nucleic acids research 20050602 10

Pseudouridine (Psi) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Ps ...[more]

PMID: 15933208

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Project description:Telomeres are protective repeats of TTAGGG sequences located at the end of human chromosomes. They are essential to maintain chromosomal integrity and genome stability. Telomerase is a ribonucleoprotein complex containing an internal RNA template (hTR) and a catalytic subunit (hTERT). The human hTR gene consists of three major domains; among them the H/ACA domain is essential for telomere biogenesis. H/ACA ribonucleoprotein (RNP) complex is composed of four evolutionary conserved proteins, including dyskerin (encoded by DKC1 gene), NOP10, NHP2 and GAR1. In this study, we have evaluated the expression profile of the H/ACA RNP complex genes: DKC1, NOP10, NHP2 and GAR1, as well as hTERT and hTR mRNA levels, in patients with chronic lymphocytic leukemia (CLL). Results were correlated with the number and type of genetic alteration detected by conventional cytogenetics and FISH (fluorescence in situ hybridization), IGHV (immunoglobulin heavy chain variable region) mutational status, telomere length (TL) and clinico-pathological characteristics of patients. Our results showed significant decreased expression of GAR1, NOP10, DKC1 and hTR, as well as increased mRNA levels of hTERT in patients compared to controls (p≤0.04). A positive correlation between the expression of GAR1-NHP2, GAR1-NOP10, and NOP10-NHP2 (p<0.0001), were observed. The analysis taking into account prognostic factors showed a significant increased expression of hTERT gene in unmutated-IGHV cases compared to mutated-CLL patients (p = 0.0185). The comparisons among FISH groups exhibited increased expression of DKC1 in cases with two or more alterations with respect to no abnormalities, trisomy 12 and del13q14, and of NHP2 and NOP10 compared to those with del13q14 (p = 0.03). The analysis according to TL showed a significant increased expression of hTERT (p = 0.0074) and DKC1 (p = 0.0036) in patients with short telomeres compared to those with long TL. No association between gene expression and clinical parameters was found. Our results suggest a role for these telomere associated genes in genomic instability and telomere dysfunction in CLL.

Dataset Information

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

Publications

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

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