The non-snRNP factors are also necessary for splicing, of which serine/arginine (SR)-rich proteins were considered as markers of splicing speckles (25). We determined whether p.Pro315Leu could affect the pattern of splicing speckles using SC-35, a non-snRNP component, as a marker (25). We performed immunofluorescence and Q-PCR on cells carrying either p.Pro315Leu or WT allele. By confocal microscopy, we found the level of endogenous SC-35 is dramatically increased with a diffused pattern in hPrp4Pro315Leu fibroblasts, whereas in control fibroblasts the SC-35 showed a distinctly speckled pattern (Fig. 4B and C). Consistently, Q-PCR revealed that the expression levels of SRSF1 (encoding SF2/ASF; NM_006924.4), another non-snRNP component and SRSF2 (encoding SC-35; NM_003016.4) were significantly increased in hPrp4Pro315Leu fibroblasts compared with WT fibroblasts (Fig. 4A). Similar changes in the levels and distribution of SC-35 were also observed in ARPE19 and 293T cells transfected with hPrp4Pro315Leu, but not in those transfected with hPrp4WT (Fig. 4D–G), indicating that the p.Pro315Leu variant of PRPF4 affected the splicing machinery.
ExperimentName: Altered pattern of splicing speckles in patient fibroblasts harboring a heterozygous p.Pro315Leu variant in PRPF4
PatientCell: CL:0000057 (fibroblast)
GeneFunction: GO:0000398 (mRNA splicing, via spliceosome)
GeneAlteration: Cells harbor a heterozygous p.Pro315Leu variant in PRPF4.
FunctionalEvidence: Patient cells harboring the variant exhibit a much higher level of endogenous marker of splicing speckles (SC-35) with a diffused distribution rather than a pattern of distinct speckles (Figures 4B and 4C). RT-PCR showed increased expression of SRSF1 and SRSF2 as well (Figure 4A). Collectively, the patient cells harboring the variant show molecular evidence of disrupted splicing.
FAVariants: The model harbors the NM_001244926.2(PRPF4):c.941C>T (p.Pro314Leu) variant in the heterozygous state (ClinVar ID: 143057).