Abstract
Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.
Highlights
Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells
Pre-mRNA splicing reaction occurs in spliceosome which is composed of 5 small nuclear ribonucleoproteins, U1, U2, U4/U6, U5, and a large number of accessory protein factors[13]
In Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system, a short guide RNA contains about a 20 nt sequence and is capable of recognizing the targeted site followed by a protospacer adjacent motif (PAM) which recruits Cas[9] to the targeted genome and induces the formation of site-specific double-stranded breaks (DSBs)
Summary
Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. We utilize the CRISPR/Cas[9] system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. Gene expression profiling by RNASeq demonstrated that RP associated genes, Fscn[2] and Bbs[2], were down-regulated in the mutated cells. A group of spliceosome genes, including PRPF36, PRPF87, PRPF318, RP99, SNRNP20010, PRPF611, and PRPF412, are ubiquitously expressed and involved in the pre-mRNA splicing process. Underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown due to lack of appropriate disease model in vitro or in vivo. Previous studies have shown that this system could modify genome editing in eukaryotic cells with high efficiency[19,20,21,22,23,24]
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