RESUMO
The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.
Assuntos
Sítios de Splice de RNA , Retinose Pigmentar , Spliceossomos , Humanos , Spliceossomos/genética , Spliceossomos/metabolismo , Proteômica , Splicing de RNA/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Mensageiro/metabolismo , Mutação , DNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes-causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease.