18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site

Optimization of Ribosome Structure and Function by rRNA Base Modification

BACKGROUND: Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. This work investigates how ribosome structure and function are affected by rRNA base modification. The prevailing view is that rRNA base modifications serve to fine tune ribosome structure and function. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, yeast strains deficient in rRNA modifications in the ribosomal peptidyltransferase center were monitored for changes in and translational fidelity. These studies revealed allele-specific sensitivity to translational inhibitors, changes in reading frame maintenance, nonsense suppression and aa-tRNA selection. Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay. rRNA chemical analyses of one of these mutants identified structural changes in five specific bases associated with the ribosomal A-site. CONCLUSIONS/SIGNIFICANCE: Together, the data suggest that modification of these bases fine tune the structure of the A-site region of the large subunit so as to assure correct positioning of critical rRNA bases involved in aa-tRNA accommodation into the PTC, of the eEF-1A•aa-tRNA•GTP ternary complex with the GTPase associated center, and of the aa-tRNA in the A-site. These findings represent a direct demonstration in support of the prevailing hypothesis that rRNA modifications serve to optimize rRNA structure for production of accurate and efficient ribosomes

Mouse dyskerin mutations affect accumulation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing

Dyskerin is a nucleolar protein present in small nucleolar ribonucleoprotein particles that modify specific uridine residues of rRNA by converting them to pseudouridine. Dyskerin is also a component of the telomerase complex. Point mutations in the human gene encoding dyskerin cause the skin and bone marrow failure syndrome dyskeratosis congenita (DC). To test the extent to which disruption of pseudouridylation or telomerase activity may contribute to the pathogenesis of DC, we introduced two dyskerin mutations into murine embryonic stem cells. The A353V mutation is the most frequent mutation in patients with X-linked DC, whereas the G402E mutation was identified in a single family. The A353V, but not the G402E, mutation led to severe destabilization of telomerase RNA, a reduction in telomerase activity, and a significant continuous loss of telomere length with increasing numbers of cell divisions during in vitro culture. Both mutations caused a defect in overall pseudouridylation and a small but detectable decrease in the rate of pre-rRNA processing. In addition, both mutant embryonic stem cell lines showed a decrease in the accumulation of a subset of H/ACA small nucleolar RNAs, correlating with a significant decrease in site-specific pseudouridylation efficiency. Interestingly, the H/ACA snoRNAs decreased in the G402E mutant cell line differed from those affected in A353V mutant cells. Hence, our findings show that point mutations in dyskerin may affect both the telomerase and pseudouridylation pathways and the extent to which these functions are altered can vary for different mutations