Identification and functional characterization of a novel splicing mutation in RP gene PRPF31

A six-generation Chinese family with autosomal dominant retinitis pigmentosa (adRP) was identified and characterized. Genome-wide linkage analysis linked the family to markers D19S601 to D19S605, which span the PRPF31 gene on chromosome 19q13.33-13.43 (RP11) (LOD = 5.03). Direct DNA sequence analysis identified a novel splicing mutation (IVS1+1G>T) in affected family members and carriers, but not in unaffected family members and 200 normal controls. The splicing mutation occurs at the splicing donor of intron 1. Real time PCR with lymphoblastoid RNA samples from family members showed that in comparison to normal family members, the splicing mutation reduced the expression level of the PRPF31 mRNA by 57% in symptomatic patients and by 28% in clinically asymptomatic carriers. Our studies identify a novel splicing mutation in PRPF31 associated with adRP and suggest that the penetrance of RP11 mutations may be correlated with the expression level of the PRPF31 mRNA.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

A DExH/D-box protein coordinates the two steps of splicing in a group I intron

Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast has shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β occurs with only two of these co-factors and, furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5β splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the “functional-timing” of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the “upstream” activity of other co-factors.