Retinitis pigmentosa (RP) is a progressive, inherited retinal degeneration disease which can be caused by mutations in any of 50 genes in a person, and it eventually will lead to the person becoming blind. RP can be inherited as either a dominant trait, a recessive trait, or it could be an X-linked trait (meaning it is a trait only tied to the X chromosome).
The tri-snRNP complex RNA splice specifically has a role in the U4/U6.U5 complex. Introns are removed through the interactions that lead to snRNP subcomplexes being assembled and disassembled. The tri-snRNP is formed by the interaction of the U4/U6 snRNP’s and U5, which is a process that is controlled by the splicing factors PRPF31 and PRPF6. The reason that the researchers specifically searched for mutations in the PRPF6 splicing factor is because it binds to 4 of the 5 tri-snRNP proteins involved in adRP (the center of the particle), therefore they believed it would be a good gene to screen for the RP mutation.
In the paper, it stated that the researchers specifically looked for the R729W mutation in the PRPF6 subcomplex because there was a missense variation in exon 16, codon 729 which caused arginine to be substituted for tryptophan. It is a variant that affected a PRPF6 residue that was found to be consistent across all eukaryotic kingdoms (with the exception of insects). This arginine 729 is a part of one the HAT repeats that are in the C-terminal part of PRPF6. Interestingly, the mutation was found in the chromosome of a patient who had a history of RP in his family, but there were over a thousand other chromosomes from subjects who were ethnically similar who did not have the mutation. More genetic screening was performed, and it was found that the affected brother of the patient had the same DNA change.
The conclusion from the pedigree analysis was that over three to four generations, there is a dominant inheritance of RP. Pictures were taken of patients 001 through 173’s right eyes at 37 years old and 55 years old, and it showed progressively weaker responses to light and the overall progression of RP.
The reason researchers isolated lymphoblast cells from individuals with and without the disease was to learn more about the pathogenicity of the DNA change, and to learn a little bit more about the consequences of the missense. The amount of the tri-snRNP, and what it is made of, is affected by mutations in the splicing factor genes that are associated with adRP. Ultimately, this leads to defects in its functions. Defects in the function of tri-snRNP is detrimental to the ability to excise introns of specific pre-mRNA substrates. They discovered that cellular localization of endogenous PRPF6 occurred after there was transformation with the Epstein-Barr virus. It was also found that the nucleoplasm and splicing speckles demonstrated localization in all of the PRPF6 cell lines. The accumulation of PRPF6 that was shown were later given the name Cajal bodies, which were absent in the control cells.
GFP-tagged PRPF6 mutants and GFP-tagged PRPF6 normal proteins were tagged to gain a deeper understanding of the process. PCR was used to multiply cDNA from a control human’s lymphoblast, and it showed the PRPF6 sequence at the 3’ end, but at the 5’ end it also had restriction sites for the EcoRI and KpnI endonucleases which were artificially introduced. By using the artificially introduced restriction sites to clone a plasmid, they discovered the mutant fusions formed the same exact phenotype as the cells that naturally contained p.Arg729Trp. This demonstrated two things. First of all, it demonstrated that mislocalized PRPF6 only relate to mutated proteins. The second thing it demonstrated was that, in HeLa cells, endogenous functional copies of PRPF6 cannot compliment that phenotype.
As previously stated, people who have mutations in the splicing factors PRPF31, PRPF3 and PRPF8 are unable to effectively excise introns from pre-mRNA. The two cell lines that had the p.Arg729Trp showed an inability to completely splice four of the five introns. They discovered that PRPF6 is the sixth splicing factor that’s involved in autosomal-dominant RP.