Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.

Cross-species single-cell landscapes identify the pathogenic gene characteristics of inherited retinal diseases.

Introduction: Inherited retinal diseases (IRDs) affect ∼4.5 million people worldwide. Elusive pathogenic variants in over 280 genes are associated with one or more clinical forms of IRDs. It is necessary to understand the complex interaction among retinal cell types and pathogenic genes by constructing a regulatory network. In this study, we attempt to establish a panoramic expression view of the cooperative work in retinal cells to understand the clinical manifestations and pathogenic bases underlying IRDs. Methods: Single-cell RNA sequencing (scRNA-seq) data on the retinas from 35 retina samples of 3 species (human, mouse, and zebrafish) including 259,087 cells were adopted to perform a comparative analysis across species. Bioinformatic tools were used to conduct weighted gene co-expression network analysis (WGCNA), single-cell regulatory network analysis, cell-cell communication analysis, and trajectory inference analysis. Results: The cross-species comparison revealed shared or species-specific gene expression patterns at single-cell resolution, such as the stathmin family genes, which were highly expressed specifically in zebrafish Müller glias (MGs). Thirteen gene modules were identified, of which nine were associated with retinal cell types, and Gene Ontology (GO) enrichment of module genes was consistent with cell-specific highly expressed genes. Many IRD genes were identified as hub genes and cell-specific regulons. Most IRDs, especially the retinitis pigmentosa (RP) genes, were enriched in rod-specific regulons. Integrated expression and transcription regulatory network genes, such as congenital stationary night blindness (CSNB) genes GRK1, PDE6B, and TRPM1, showed cell-specific expression and transcription characteristics in either rods or bipolar cells (BCs). IRD genes showed evolutionary conservation (GNAT2, PDE6G, and SAG) and divergence (GNAT2, MT-ND4, and PDE6A) along the trajectory of photoreceptors (PRs) among species. In particular, the Leber congenital amaurosis (LCA) gene OTX2 showed high expression at the beginning of the trajectory of both PRs and BCs. Conclusion: We identified molecular pathways and cell types closely connected with IRDs, bridging the gap between gene expression, genetics, and pathogenesis. The IRD genes enriched in cell-specific modules and regulons suggest that these diseases share common etiological bases. Overall, mining of interspecies transcriptome data reveals conserved transcriptomic features of retinas across species and promising applications in both normal retina anatomy and retina pathology.

The splicing factor DHX38 enables retinal development through safeguarding genome integrity.

DEAH-Box Helicase 38 (DHX38) is a pre-mRNA splicing factor and also a disease-causing gene of autosomal recessive retinitis pigmentosa (arRP). The role of DHX38 in the development and maintenance of the retina remains largely unknown. In this study, by using the dhx38 knockout zebrafish model, we demonstrated that Dhx38 deficiency causes severe differentiation defects and apoptosis of retinal progenitor cells (RPCs) through disrupted mitosis and increased DNA damage. Furthermore, we found a significant accumulation of R-loops in the dhx38-deficient RPCs and human cell lines. Finally, we found that DNA replication stress is the prerequisite for R-loop-induced DNA damage in the DHX38 knockdown cells. Taken together, our study demonstrates a necessary role of DHX38 in the development of retina and reveals a DHX38/R-loop/replication stress/DNA damage regulatory axis that is relatively independent of the known functions of DHX38 in mitosis control.