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R354X mutation in PRPF31 affects its capacity in inflammation response and splicing

Xiaoqiang Xiao* Fang Deng, Shaofen Huang

Pre-mRNA Processing Factor 31(PRPF31) is a key component of RNA splicing and a disease-causing gene of Retinitis Pigmentosa (RP). Previously, we found that the nonsense mutation R354X in PRPF31 induces RP in a Chinese RP family. In order to investigate the underlining molecular mechanisms of RP pathogenesis induced by this mutation, we generated cell lines stably expressing R354X mutant, Wild Type (WT) of PRPF31 and corresponding empty vector using HEK293T cells ,the resulting cell lines were used for Long non-coding RNA sequencing (LncRNA-sequencing). The results of LncRNA sequencing showed that, comparing to WT, R354X mutation changed the expression and splicing of coding and non-coding transcripts. Interestingly, in HEK293T and APRE-19 cells, inflammationassociated genes such as IFI6, OAS3and STAT3, enhanced their expression in response to the overexpression of WT PRPF31; however, in R354X mutation cells, those gene’s expression remained basal levels. Moreover, increased H2AFX expression and attenuated growth capacity were found in cells expressing R354X PRPF31 in HEK293T. In contrast with WT, R354X mutant showed varied splicing model on Di Hydro Folate Reductase (DHFR) in HEK293T. During IP analysis, we found that R354X mutant reduced its binding with CPSF1 and SORBS1 mRNAs in ARPE-19, and its binding with CTNNBL1 was also interfered in ARPE-19 cells. On the other hand, the R354X mutant also increased the level of transcripts read-through. Taken together, R354X mutation in PRPF31 affected cell survival, changed gene’s expression and splicing. Those findings indicate that inflammation and oxidation might contribute the pathogenesis of RP induced by the R354X mutation.