Second, binding of PRPF4 to its CX-4945 interactor PRPF3 was abrogated by the p.R192H amino acid exchange. Third, variant PRPF4 failed to integrate into the tri-snRNP in human cell lines and zebrafish embryos. Together, these findings indicate that PRPF4 haploinsufficiency may contribute to RP. Furthermore, they underline the role of trisnRNP splicing factors as mutational hotspots in RP and demonstrate the feasibility of a direct analysis of these candidate genes in RP patients. In this study, we describe the identification of an isolated RP patient that carries a heterozygous missense variant in the gene encoding the tri-snRNP splicing factor PRPF4. We show that this variant disrupts the binding of PRPF4 to its interactor PRPF3 in vitro. This leads to an impaired integration into spliceosomal particles and a loss of physiological function in vivo. PRPF4 has been shown previously to form a very stable ternary complex with PRPF3 and the peptidyl-prolyl isomerase PPIH. Within this ternary complex, PRPF4 acts as a bridging factor by contacting PPIH via its N-terminal part and PRPF3 via its central and C-terminal part. Consistent with this mapping is our observation that the p.R192H mutation affected PRPF3 binding but not the association with PPIH. This mode of binding suggests that p.R192H not only causes a defective integration of PRPF4 into the tri-snRNP, but that it might also lead to the concomitant loss of PPIH, a protein necessary for efficient splicing. Remarkably, a similar mechanism has been proposed for an RPmutant form of PRPF31 that is thought to sequester its interactor PRPF6 out of the tri-snRNP, and an altered composition of the tri-snRNP has also been described for RP-causing missense mutations in PRPF8. Our biochemical data strongly indicate that the p.R192H variant leads to the production of non-functional protein and thus represents a functional null allele. However, patient RP-106 has two daughters, one of which was found to have inherited the c.575G.A variant. Both daughters underwent detailed ophthalmological investigation and did not show signs of retinal degeneration. While the genetic study presented here thus does not allow to conclude whether PRPF4 is causally linked to RP, additional evidence indicates that this is indeed the case: Our data obtained in zebrafish showed that the sub-lethal knockdown of Prpf4 leads to a retina-specific phenotype similar to that of RP. The data presented here shows that wildtype Prpf4, but not a variant corresponding to p.R192H can rescue such Prpf4 deficiency. Further support for a link between PRPF4 deficiency and RP comes from a recent study where PRPF4 mutations were found to cause adRP in a Chinese cohort. Also, a potentially pathogenic missense variant of PRPF4 was reported in two German siblings with RP. Although this latter study failed to demonstrate a clear association between PRPF4 and RP in North American and European populations, these data indicate that rare defects in PRPF4 might cause RP in a limited subset of patients.