The C-terminal extension of dyskerin is a dyskeratosis congenita mutational hotspot that modulates interaction with telomerase RNA and subcellular localization.

Dyskerin is a component of the human telomerase complex and is involved in stabilizing the human telomerase RNA (hTR). Many mutations in the DKC1 gene encoding dyskerin are found in X-linked dyskeratosis congenita (X-DC), a premature aging disorder and other related diseases. The C-terminal extension (CTE) of dyskerin contributes to its interaction with the molecular chaperone SHQ1 during the early stage of telomerase biogenesis. Disease mutations in this region were proposed to disrupt dyskerin-SHQ1 interaction and destabilize dyskerin, reducing hTR levels indirectly. However, biochemical evidence supporting this hypothesis is still lacking. In addition, the effects of many CTE disease mutations on hTR have not been examined. In this study, we tested eight dyskerin CTE variants and showed that they failed to maintain hTR levels. These mutants showed slightly reduced but not abolished interaction with SHQ1, and caused defective binding to hTR. Deletion of the CTE further reduced binding to hTR, and perturbed localization of dyskerin to the Cajal bodies and the nucleolus, and the interaction with TCAB1 as well as GAR1. Our findings suggest impaired dyskerin-hTR interaction in cells as a previously overlooked mechanism through which dyskerin CTE mutations cause X-DC and related telomere syndromes.

RNA-guided isomerization of uridine to pseudouridine--pseudouridylation.

Box H/ACA ribonucleoproteins (RNPs), each consisting of one unique guide RNA and 4 common core proteins, constitute a family of complex enzymes that catalyze, in an RNA-guided manner, the isomerization of uridines to pseudouridines (Ψs) in RNAs, a reaction known as pseudouridylation. Over the years, box H/ACA RNPs have been extensively studied revealing many important aspects of these RNA modifying machines. In this review, we focus on the composition, structure, and biogenesis of H/ACA RNPs. We explain the mechanism of how this enzyme family recognizes and specifies its target uridine in a substrate RNA. We discuss the substrates of box H/ACA RNPs, focusing on rRNA (rRNA) and spliceosomal small nuclear RNA (snRNA). We describe the modification product Ψ and its contribution to RNA function. Finally, we consider possible mechanisms of the bone marrow failure syndrome dyskeratosis congenita and of prostate and other cancers linked to mutations in H/ACA RNPs.

Recurrent deletion of 3p13 targets multiple tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion-positive prostate cancers.

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1-specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG(+) cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG(+) tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG(+) cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over-expression of FOXP1, RYBP and SHQ1 resulted in decreased colony-formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG(+) prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors.

In silico characterization and identification of compound heterozygous variants in H/ACA Ribonucleoprotein Assembly Factor (SHQ1) from Indian population.

Background: H/ACA small nucleolar ribonucleoproteins (snoRNP) form a complex with multiple proteins to accomplish the pseudouridylation of rRNA. The assembly of H/ACA small nucleolar ribonucleoproteins (snoRNP) is initiated by H/ACA ribonucleoprotein Assembly factor, that is, SHQ1. Mutations in SHQ1 have been reported to cause two disorders namely, dystonia-35 childhood onset (OMIM*619921) and neurodevelopmental disorder with seizures and dystonia (OMIM*619922), both of which are inherited in an autosomal recessive manner. Considering the high genetic and clinical diversity of SHQ1-related clinical features and the importance of SHQ1 in the assembly of the H/ACA snoRNP complex, it is important to take a systematic approach to delineate the genetic diagnosis and impact of mutations on protein structure and stability. Methods: Whole exome sequencing followed by Sanger validation was performed in an individual with the clinical features of neurodevelopmental disorder with seizures and dystonia (OMIM*619922). Protein modeling studies of all the reported SHQ1 variants to date were performed using freely available web servers Interactive Tree of Life, String, BioGrid, ShinyGO, DAVID, and Pathvix. Protein structures were visualized using Pymol. Results and Discussion: We identified compound heterozygous variants, one known frameshift deletion c. 828_831del, p.(Asp277Serfs*27) and the other novel missense variant c. 1157A>C, p.(Tyr386Ser) found in an individual with neurodevelopmental disorder, seizures, movement disorder, and hypomyelination leukodystrophy on neuroimaging. Protein-interactome studies identified potential genetic interactors that include GAR1, NAF1, TRUB1, UTP15, DKC1, NOP10, NPHOSPH 10, KRR1, NOP58, NOP56, FBL, RRP9, NHP2, RUVBL1, and RUVBL2. Ribosome biogenesis in eukaryotes, RNA polymerase, RNA transport, spliceosome, ribosome, cytosolic DNA-sensing pathway, DNA replication, mismatch repair, base excision repair, nucleotide excision repair, and basal transcription factors process were identified as the linked pathways with the prioritized genes. Conclusion: In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

ß-Sitosterol attenuates anlotinib resistance in non-small cell lung cancer cells by inhibiting miR-181a-3p/SHQ1 signaling.

Anlotinib is used for the treatment of advanced non-small cell lung cancer; however, the emergence of drug resistance limits its clinical application. ß-sitosterol may also be used to treat lung cancer, but there have been no studies evaluating ß-sitosterol against anlotinib-resistant lung cancer. The purpose of this study was to determine the mechanism by which ß-sitosterol enhances the sensitivity of lung cancer cells to anlotinib. A549 cells were treated with different concentrations of anlotinib to generate anlotinib-resistant cells (A549/anlotinib cells). miR-181a-3p mimics were transfected into A549/anlotinib cells. A549 and A549/anlotinib cells were treated with ß-sitosterol at various concentrations. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. Apoptosis was assessed by flow cytometry. Real-time quantitative PCR was used to measure the expression of miR-181a-3p. The interaction of miR-181a-3p with the H/ACA ribonucleoprotein assembly factor (SHQ1) was predicted using the miRDB and TargetScan Human databases and verified with a luciferase reporter assay. The expression of SHQ1, activating transcription factor 6 (ATF6), and glucose-regulated protein 78 (GRP78) were measured by western blot analysis. ß-Sitosterol effectively suppressed A549/anlotinib cell proliferation and promoted apoptosis. SHQ1 is a downstream target of miR-181a-3p. The expression of miR-181a-3p was inhibited; however, SHQ1 expression was increased by ß-sitosterol treatment of A549/anlotinib cells. The inhibition of SHQ1, ATF6, and GRP78 protein expression by ß-sitosterol in A549/anlotinib cells was rescued by increased miR-181a-3p. ß-Sitosterol markedly promotes anlotinib-resistant A549 cell apoptosis and inhibits cell proliferation by activating SHQ1/UPR signaling through miR-181a-3p inhibition.

Human SHQ1 variants R335C and A426V lead to severe ribosome biogenesis defects when expressed in yeast.

SHQ1 is an essential chaperone that binds the pseudouridine synthase dyskerin in the cytoplasm and escorts the enzyme to the nucleus, where dyskerin is assembled into small nucleolar RNPs (snoRNPs) of the H/ACA class. These particles carry out pseudouridine formation in ribosomal RNAs (rRNAs) and participate in maturation of rRNA precursors (pre-rRNAs). Variants of human SHQ1 have been linked to neurodevelopmental deficiencies; here we focused on two compound heterozygous mutations identified in a child showing a severe neurological disorder comprising cerebellar degeneration. To investigate the molecular defects caused by mutations R335C and A426V we used a conditional yeast strain that can be depleted of the endogenous Shq1 protein while constitutively expressing human SHQ1 (wild-type or variants). Although wild-type SHQ1 complemented the Shq1-depleted strain, cells expressing variant R335C could not support growth, and cells expressing variant A426V were temperature-sensitive. When shifted to restrictive conditions, yeast cells progressively lost H/ACA snoRNAs and accumulated unprocessed pre-rRNAs, which led to reduced production of ribosomes. Levels of Cbf5 (yeast homologue of dyskerin) were decreased in yeast cells expressing SHQ1 variants under restrictive conditions. Immunoprecipitation experiments revealed that interaction of Cbf5 with SHQ1 variants was weakened but not abolished, and yeast two-hybrid assays showed that mutation R335C is more deleterious than mutation A426V. Our data provide additional evidence for the critical role of SHQ1 in chaperoning the pseudouridine synthase dyskerin, and how its inadequate function has detrimental consequences on the production of H/ACA snoRNPs and ribosomes.