Spliceosomic dysregulation in pancreatic cancer uncovers splicing factors PRPF8 and RBMX as novel candidate actionable targets.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, characterized by late diagnosis and poor treatment response. Surgery is the only curative approach, only available to early-diagnosed patients. Current therapies have limited effects, cause severe toxicities, and minimally improve overall survival. Understanding of splicing machinery alterations in PDAC remains incomplete. Here, we comprehensively examined 59 splicing machinery components, uncovering dysregulation in pre-mRNA processing factor 8 (PRPF8) and RNA-binding motif protein X-linked (RBMX). Their downregulated expression was linked to poor prognosis and malignancy features, including tumor stage, invasion and metastasis, and associated with poorer survival and the mutation of key PDAC genes. Experimental modulation of these splicing factors in pancreatic cancer cell lines reverted their expression to non-tumor levels and resulted in decreased key tumor-related features. These results provide evidence that the splicing machinery is altered in PDAC, wherein PRPF8 and RBMX emerge as candidate actionable therapeutic targets.

Altered splicing machinery in lung carcinoids unveils NOVA1, PRPF8 and SRSF10 as novel candidates to understand tumor biology and expand biomarker discovery.

BACKGROUND: Lung neuroendocrine neoplasms (LungNENs) comprise a heterogeneous group of tumors ranging from indolent lesions with good prognosis to highly aggressive cancers. Carcinoids are the rarest LungNENs, display low to intermediate malignancy and may be surgically managed, but show resistance to radiotherapy/chemotherapy in case of metastasis. Molecular profiling is providing new information to understand lung carcinoids, but its clinical value is still limited. Altered alternative splicing is emerging as a novel cancer hallmark unveiling a highly informative layer. METHODS: We primarily examined the status of the splicing machinery in lung carcinoids, by assessing the expression profile of the core spliceosome components and selected splicing factors in a cohort of 25 carcinoids using a microfluidic array. Results were validated in an external set of 51 samples. Dysregulation of splicing variants was further explored in silico in a separate set of 18 atypical carcinoids. Selected altered factors were tested by immunohistochemistry, their associations with clinical features were assessed and their putative functional roles were evaluated in vitro in two lung carcinoid-derived cell lines. RESULTS: The expression profile of the splicing machinery was profoundly dysregulated. Clustering and classification analyses highlighted five splicing factors: NOVA1, SRSF1, SRSF10, SRSF9 and PRPF8. Anatomopathological analysis showed protein differences in the presence of NOVA1, PRPF8 and SRSF10 in tumor versus non-tumor tissue. Expression levels of each of these factors were differentially related to distinct number and profiles of splicing events, and were associated to both common and disparate functional pathways. Accordingly, modulating the expression of NOVA1, PRPF8 and SRSF10 in vitro predictably influenced cell proliferation and colony formation, supporting their functional relevance and potential as actionable targets. CONCLUSIONS: These results provide primary evidence for dysregulation of the splicing machinery in lung carcinoids and suggest a plausible functional role and therapeutic targetability of NOVA1, PRPF8 and SRSF10.

PRPF8 controls alternative splicing of PIRH2 to modulate the p53 pathway and survival of human ESCs.

Human embryonic stem cells (hESCs) have great potential for developmental biology and regenerative medicine. However, extensive apoptosis often occurs when hESCs respond to various stresses or injuries. Understanding the molecular control and identifying new factors associated with hESC survival are fundamental to ensure the high quality of hESCs. In this study, we report that PRPF8, an RNA spliceosome component, is essential for hESC survival. PRPF8 knockdown (KD) induces p53 protein accumulation and activates the p53 pathway, leading to apoptosis in hESCs. Strikingly, silencing of p53 rescues PRPF8 KD-induced apoptosis, indicating that PRPF8 KD triggers hESC apoptosis through activating the p53 pathway. In search for the mechanism by which p53 pathway is activated by PRPF8 KD, we find that PRPF8 KD alters alternative splicing of many genes, including PIRH2 which encodes an E3 ubiquitin ligase of p53. PIRH2 has several isoforms such as PIRH2A, PIRH2B, and PIRH2C. Intriguingly, PRPF8 KD specifically increases the transcript level of the PIRH2B isoform, which lacks a RING domain and E3 ligase activity. Functionally, PIRH2B KD partially rescues the reduction in cell numbers and upregulation of P21 caused by PRPF8 KD in hESCs. The finding suggests that PRPF8 controls alternative splicing of PIRH2 to maintain the balance of p53 pathway activity and survival of hESCs. The PRPF8/PIRH2/p53 axis identified here provides new insights into how p53 pathway and hESC survival are precisely regulated at multiple layers, highlighting an important role of posttranscriptional machinery in supporting hESC survival.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8.

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2-PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast.

Cilia regeneration requires an RNA splicing factor from the ciliary base.

Cilia are microtubule-based organelles projected from most eukaryotic cell surfaces performing cell motility and signaling. Several previously recognized non-ciliary proteins play crucial roles in cilium formation and function. Here, we provide additional evidence that the Caenorhabditis elegans RNA splicing factor PRP-8/PRPF8 regulates ciliogenesis and regeneration from the ciliary base. Live imaging of GFP knock-in animals reveals that the endogenous PRP-8 localizes in the nuclei and the ciliary base. A weak loss-of-function allele of prp-8 affects ciliary structure but with little impact on RNA splicing. Conditional degradation of PRP-8 within ciliated sensory neurons showed its direct and specific roles in cilium formation. Notably, the penetrance of ciliary defects correlates with the reduction of PRP-8 at the ciliary base but not nuclei, and sensory neurons regenerated cilia accompanying PRP-8 recovery from the ciliary base rather than the nuclei. We suggest that PRP-8 at the ciliary base contributes to cilium formation and regeneration.

Cytogenetic and Genetic Abnormalities with Diagnostic Value in Myelodysplastic Syndromes (MDS): Focus on the Pre-Messenger RNA Splicing Process.

Myelodysplastic syndromes (MDS) are considered to be diseases associated with splicing defects. A large number of genes involved in the pre-messenger RNA splicing process are mutated in MDS. Deletion of 5q and 7q are of diagnostic value, and those chromosome regions bear the numbers of splicing genes potentially deleted in del(5q) and del(7q)/-7 MDS. In this review, we present the splicing genes already known or suspected to be implicated in MDS pathogenesis. First, we focus on the splicing genes located on chromosome 5 (HNRNPA0, RBM27, RBM22, SLU7, DDX41), chromosome 7 (LUC7L2), and on the SF3B1 gene since both chromosome aberrations and the SF3B1 mutation are the only genetic abnormalities in splicing genes with clear diagnostic values. Then, we present and discuss other splicing genes that are showing a prognostic interest (SRSF2, U2AF1, ZRSR2, U2AF2, and PRPF8). Finally, we discuss the haploinsufficiency of splicing genes, especially from chromosomes 5 and 7, the important amplifier process of splicing defects, and the cumulative and synergistic effect of splicing genes defects in the MDS pathogenesis. At the time, when many authors suggest including the sequencing of some splicing genes to improve the diagnosis and the prognosis of MDS, a better understanding of these cooperative defects is needed.

The role of splicing factor PRPF8 in breast cancer.

BACKGROUND: Alternative splicing is a mechanism to produce different proteins with diverse functions from one gene. Many splicing factors play an important role in cancer progression. PRPF8 is a core protein component of the spliceosome complex, U4/U6-U5 tri-snRNP. OBJECTIVE: However, PRPF8 involved in mRNA alternative splicing are rarely included in the prognosis. METHODS: We found that PRPF8 was expressed in all examined cancer types. Further analyses found that PRPF8 expression was significantly different between the breast cancer and paracancerous tissues. RESULTS: Survival analyses showed that PRPF8-high patients had a poor prognosis, and the expression of PRPF8 is associated with distant metastasis-free survival (DMFS) and post progression survival (PPS). Gene Set Enrichment Analysis (GSEA) has revealed that PRPF8 expression is correlated with TGF-ß, JAK-STAT, and cell cycle control pathways. Consistent with these results, upon PRPF8 silencing, the growth of MCF-7 cells was reduced, the ability of cell clone formation was weakened, and p⁢21 expression was increased. CONCLUSIONS: These results have revealed that PRPF8 is a significant factor for splicing in breast cancer progression.

Landscape of pathogenic variants in six pre-mRNA processing factor genes for retinitis pigmentosa based on large in-house data sets and database comparisons.

PURPOSE: Variants in six genes encoding pre-mRNA processing factors (PRPFs) are a common cause of autosomal dominant retinitis pigmentosa (ADRP). This study aims to determine the characteristics of potential pathogenic variants (PPVs) in the six genes. METHODS: Variants in six PRPF genes were identified from in-house exome sequencing data. PPVs were identified based on comparative bioinformatics analysis, clinical phenotypes and the ACMG/AMP guidelines. The features of PPVs were revealed by comparative analysis of in-house data set, gnomAD and previously published literature. RESULTS: Totally, 36 heterozygous PPVs, including 19 novels, were detected from 45 families, which contributed to 4.4% (45/1019) of RP cases. These PPVs were distributed in PRPF31 (17/45, 37.8%), SNRNP200 (12/45, 26.7%), PRPF8 (10/45, 22.2%) and PRPF3 (6/45, 13.3%) but not in PRPF6 or PRPF4. Different types of PPVs were predominant in different PRPF genes, such as loss-of-function variants in PRPF31 and missense variants in the five remaining genes. The clustering of PPVs in specific regions was observed in SNRNP200, PRPF8 and PRPF3. The pathogenicity for certain classes of variants in these genes, such as loss-of-function variants in PRPF6 and missense variants in PRPF31 and PRPF4, requires careful consideration and further validation. The predominant fundus changes were early macular involvement, widespread RPE atrophy and pigmentation in the mid- and far-peripheral retina. CONCLUSION: Systemic comparative analysis may shed light on the characterization of PPVs in these genes. Our findings provide a brief landscape of PPVs in PRPF genes and the associated phenotypes and emphasize the careful classification of pathogenicity for certain types of variants that warrant further characterization.

Intermediate Uveitis in Retinitis Pigmentosa Associated with a Novel Homozygous Splice Site Mutation in PRPF8.

The manifestation of intermediate uveitis (IU) in patients with retinitis pigmentosa (RP) is uncommon and poses diagnostic and management challenges. In this case, we describe the clinical features and management outcomes in an RP patient with a novel homozygous splice site mutation in PRPF8. A 21-year-old male presented with unilateral decrease of vision in the right eye for 1 week. Retinal dystrophy features were present in the left eye. After 2 weeks of topical steroid therapy, near-total resolution of IU was achieved and vision improved to 20/30. Signs of (RP) were present bilaterally, with the right eye more affected than the left. Genetic testing indicated a novel homozygous c. 3061-6_3061-3del mutation in the PRPF8 gene. IU in young patients with RP can be effectively treated with a short course of topical steroids, sparing the need for systemic immunosuppressives. After the improvement in IU, the right eye showed more advanced RP changes.

BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.

Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells.

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.

Extending the phenotypic spectrum of PRPF8, PRPH2, RP1 and RPGR, and the genotypic spectrum of early-onset severe retinal dystrophy.

PURPOSE: To present the detailed retinal phenotype of patients with Leber Congenital Amaurosis/Early-Onset Severe Retinal Dystrophy (LCA/EOSRD) caused by sequence variants in four genes, either not (n = 1) or very rarely (n = 3) previously associated with the disease. METHODS: Retrospective case series of LCA/EOSRD from four pedigrees. Chart review of clinical notes, multimodal retinal imaging, electrophysiology, and molecular genetic testing at a single tertiary referral center (Moorfields Eye Hospital, London, UK). RESULTS: The mean age of presentation was 3 months of age, with disease onset in the first year of life in all cases. Molecular genetic testing revealed the following disease-causing variants: PRPF8 (heterozygous c.5804G > A), PRPH2 (homozygous c.620_627delinsTA, novel variant), RP1 (homozygous c.4147_4151delGGATT, novel variant) and RPGR (heterozygous c.1894_1897delGACA). PRPF8, PRPH2, and RP1 variants have very rarely been reported, either as unique cases or case reports, with limited clinical data presented. RPGR variants have not previously been associated with LCA/EOSRD. Clinical history and detailed retinal imaging are presented. CONCLUSIONS: The reported cases extend the phenotypic spectrum of PRPF8-, PRPH2-, RP1-, and RPGR-associated disease, and the genotypic spectrum of LCA/EOSRD. The study highlights the importance of retinal and functional phenotyping, and the importance of specific genetic diagnosis to potential future therapy.

A mitochondrial response to oxidative stress mediated by unscheduled RNA-DNA hybrids (R-loops).

How oxidative stress promotes aging-related human diseases like cancer and neurodegeneration remains unclear. Here, we discuss the origins and implications of an oxidative-stress response recently reported to destabilize the mitochondrial (mt) genome via unscheduled RNA/DNA hybrid (R-loop) accumulation, by impairing the recruitment of RNAseH1 to the regulatory regions of mtDNA.

circRNA-UBAP2 promotes the proliferation and inhibits apoptosis of ovarian cancer though miR-382-5p/PRPF8 axis.

OBJECTIVE: circular RNAs (circRNAs) have been reported to be essential regulators of multiple malignant cancers. However, the functions of circRNAs in ovarian cancer need to be further explored. The aim of our study is to explore the role of circRNA-UBAP2 in ovarian cancer and its mechanism. RESULTS: circRNA-UBAP2 was upregulated in ovarian cancer tissues and cell lines. Knockdown of circRNA-UBAP2 inhibited cell proliferation and promoted cell apoptosis, but circRNA-UBAP2 overexpressed got opposite results. In addition, circRNA-UBAP2 targeted miR-382-5p and downregulated its expression, PRPF8 was a target gene of miR-382-5p. Furthemore, circRNA-UBAP2/miR-382-5p/PRPF8 axis affected the proliferation, apoptosis and cell cycle of ovarian cancer through the mechanism of competing endogenous RNAs (ceRNA). CONCLUSION: circRNA-UBAP2 acted as a ceRNA to sponged miR-382-5p, increased the expression level of PRPF8, and prompted proliferation and inhibited apoptosis in ovarian cancer cells.

Meta-Analyses Support Previous and Novel Autism Candidate Genes: Outcomes of an Unexplored Brazilian Cohort.

Large genomic databases of neurodevelopmental disorders (NDD) are helpful resources of genomic variations in complex and heterogeneous conditions, as Autism Spectrum Disorder (ASD). We evaluated the role of rare copy number variations (CNVs) and exonic de novo variants, in a molecularly unexplored Brazilian cohort of 30 ASD trios (n = 90), by performing a meta-analysis of our findings in more than 20,000 patients from NDD cohorts. We identified three pathogenic CNVs: two duplications on 1q21 and 17p13, and one deletion on 4q35. CNVs meta-analysis (n = 8,688 cases and n = 3,591 controls) confirmed 1q21 relevance by identifying duplications in other 16 ASD patients. Exome analysis led the identification of seven de novo variants in ASD genes (SFARI list): three loss-of-function pathogenic variants in CUL3, CACNA1H, and SHANK3; one missense pathogenic variant in KCNB1; and three deleterious missense variants in ATP10A, ANKS1B, and DOCK1. From the remaining 12 de novo variants in non-previous ASD genes, we prioritized PRPF8 and RBM14. Meta-analysis (n = 13,754 probands; n = 2,299 controls) identified six and two additional patients with validated de novo variants in PRPF8 and RBM14, respectively. By comparing the de novo variants with a previously established mutational rate model, PRPF8 showed nominal significance before multiple test correction (P = 0.039, P-value adjusted = 0.079, binomial test), suggesting its relevance to ASD. Approximately 60% of our patients presented comorbidities, and the diagnostic yield was estimated in 23% (7/30: three pathogenic CNVs and four pathogenic de novo variants). Our uncharacterized Brazilian cohort with tetra-hybrid ethnic composition was a valuable resource to validate and identify possible novel candidate loci. Autism Res 2020, 13: 199-206. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: We believed that to study an unexplored autistic population, such as the Brazilian, could help to find novel genes for autism. In order to test this idea, with our limited budget, we compared candidate genes obtained from genomic analyses of 30 children and their parents, with those of more than 20,000 individuals from international studies. Happily, we identified a genetic cause in 23% of our patients and suggest a possible novel candidate gene for autism (PRPF8).

RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.

Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.

Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.

Mutation Analysis of Pre-mRNA Splicing Genes PRPF31, PRPF8, and SNRNP200 in Chinese Families with Autosomal Dominant Retinitis Pigmentosa.

BACKGROUND: To screen variants in pre-mRNA Splicing genes in 95 Chinese autosomal dominant retinitis pigmentosa (adRP) families. METHODS: Clinical examination and pedigree analysis were performed. Targeted exome sequencing (TES) and / or Sanger sequencing were performed to detect the variants in genes of Splicing factors and conduct intra-familiar segregation analysis with DNA available. In silico analysis was performed to predict pathogenicity of variants in protein level and in vitro splicing assays were performed to compare splicing variants with their corresponding wildtype about their splicing effect. RESULTS: In this study, total nine different variants were identified in PRPF31, SNRNP200, and PRPF8 respectively, including six PRPF31 variants [five novel variants 322+1G>A, c.527+2T>G, c.590T>C(p.Leu197Pro), c.1035_1036insGC (p.Pro346Argfs X18), and c.1224dupG (p.Gln409AlafsX66) plus one reported variant c.1060C>T (p.Arg354X)], a recurrent PRPF8 variant c.6930G>T (p.Arg2310Ser), two SNRNP200 variants [one heterozygous and homozygous SNRNP200 recurrent variant c.3260G>A (p.Ser1087Leu), and a reported heterozygous c.2042G>A(p.Arg681His)]. In family 20009, incomplete penetrance was observed. A novel PRPF31 missense variant c.590T>C (p.Leu197Pro) was predicted to be pathogenic in protein level via in silico analysis and in vitro splicing assay demonstrated that two novel splicing PRPF31 variants c.322+1G>A and c.527+2T>G affect splicing compared with the wildtype. CONCLUSIONS: In our studies, RP-causing variants of pre-mRNA Splicing genes (PRPF31, PRPF8 and SNRNP200) were identified in nine of the ninety-five adRP families respectively, which extend the spectra of RP variant and phenotype. And we provide the first example that SNRNP200-related RP can be caused by both heterozygous and homozygous variants of this gene.

Autosomal dominant retinitis pigmentosa-associated gene PRPF8 is essential for hypoxia-induced mitophagy through regulating ULK1 mRNA splicing.

Aged and damaged mitochondria can be selectively degraded by specific autophagic elimination, termed mitophagy. Defects in mitophagy have been increasingly linked to several diseases including neurodegenerative diseases, metabolic diseases and other aging-related diseases. However, the molecular mechanisms of mitophagy are not fully understood. Here, we identify PRPF8 (pre-mRNA processing factor 8), a core component of the spliceosome, as an essential mediator in hypoxia-induced mitophagy from an RNAi screen based on a fluorescent mitophagy reporter, mt-Keima. Knockdown of PRPF8 significantly impairs mitophagosome formation and subsequent mitochondrial clearance through the aberrant mRNA splicing of ULK1, which mediates macroautophagy/autophagy initiation. Importantly, autosomal dominant retinitis pigmentosa (adRP)-associated PRPF8 mutant R2310K is defective in regulating mitophagy. Moreover, knockdown of other adRP-associated splicing factors, including PRPF6, PRPF31 and SNRNP200, also lead to ULK1 mRNA mis-splicing and mitophagy defects. Thus, these findings demonstrate that PRPF8 is essential for mitophagy and suggest that dysregulation of spliceosome-mediated mitophagy may contribute to pathogenesis of retinitis pigmentosa.