Abstract
Post-transcriptional pseudouridylation and 2′-O-methylation of splicesomal small nuclear ribonucleic acids (snRNAs) is mediated by box H/ACA and box C/D small Cajal body (CB)-specific ribonucleoproteins (scaRNPs), respectively. The WD-repeat protein 79 (WDR79) has been proposed to interact with both classes of modification scaRNPs and target them into the CB. The box H/ACA scaRNAs carry the common CAB box motif (consensus, ugAG) that is required for both WDR79 binding and CB-specific accumulation. Thus far, no cis-acting CB-localization element has been reported for vertebrate box C/D scaRNAs. In this study, systematic mutational analysis of the human U90 and another newly identified box C/D scaRNA, mgU2-47, demonstrated that the CB-specific accumulation of vertebrate intron-encoded box C/D scaRNAs relies on GU- or UG-dominated dinucleotide repeat sequences which are predicted to form the terminal stem-loop of the RNA apical hairpin. While the loop nucleotides are unimportant, the adjacent terminal helix that is composed mostly of consecutive G.U and U.G wobble base-pairs is essential for CB-specific localization of box C/D scaRNAs. Co-immunoprecipitation experiments confirmed that the newly identified CB localization element, called the G.U/U.G wobble stem, is crucial for in vivo association of box C/D scaRNPs with WDR79.
Highlights
Eukaryotic ribosomal ribonucleic acids and spliceosomal small nuclear RNAs undergo extensive post-transcriptional 2 -O-ribose methylation and pseudouridylation during maturation [1,2]
During analysis of HeLa cellular RNAs associated with the WRD79 small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) protein [53], we have identified a novel box C/D RNA that was predicted to direct synthesis of the evolutionarily conserved Um47 2 -O-methyl-uridine in the human U2 spliceosomal small nuclear RNAs (snRNAs) [1] (Figure 1A)
Both box C/D and H/ACA RNAs are synthesized in the nucleoplasm and after nucleolytic processing and assembly with C/D and H/ACA proteins, the mature RNPs are targeted either into the nucleolus or into the Cajal body (CB), depending on their functions
Summary
Eukaryotic ribosomal ribonucleic acids (rRNAs) and spliceosomal small nuclear RNAs (snRNAs) undergo extensive post-transcriptional 2 -O-ribose methylation and pseudouridylation during maturation [1,2]. Apart from the common kink-turn motif formed by the conserved C (RUGAUGA) and D (CUGA) boxes and the flanking 5 - and 3 terminal sequences, the 2 -O-methylation guide RNAs show no overall structural conservation [19,20,21]. Both classes of guide RNAs carry short target recognition sequences which transiently base-pair to the complementary rRNA and snRNA sequences to position the selected substrate ribonucleotide at the catalytic centre of the guide RNAassociated methyltransferase (fibrillarin) or pseudouridine synthase (dyskerin) (reviewed in 22–26)
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