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Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations

Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations
Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations
Retinitis pigmentosa (RP) is the most common cause of hereditary blindness, and may occur in isolation as a non-syndromic condition or alongside other features in a syndromic presentation. Biallelic or monoallelic mutations in one of eight genes encoding pre-mRNA splicing factors are associated with non-syndromic RP. The molecular mechanism of disease remains incompletely understood, limiting opportunities for targeted treatment. Here we use CRISPR and base edited PRPF6 and PRPF31 mutant cell lines, and publicly-available data from human PRPF31+/− patient derived retinal organoids and PRPF31 siRNA-treated organotypic retinal cultures to confirm an enrichment of differential splicing of microtubule, centrosomal, cilium and DNA damage response pathway genes in these cells. We show that genes with microtubule/centrosome/centriole/cilium gene ontology terms are enriched for weak 3′ and 5′ splice sites, and that subtle defects in spliceosome activity predominantly affect efficiency of splicing of these exons. We suggest that the primary defect in PRPF6 or PRPF31 mutant cells is microtubule and centrosomal defects, leading to defects in cilium and mitotic spindle stability, with the latter leading to DNA damage, triggering differential splicing of DNA damage response genes to activate this pathway. Finally, we expand understanding of “splicing factor RP” by investigating the function of TTLL3, one of the most statistically differentially expressed genes in PRPF6 and PRPF31 mutant cells. We identify that TTLL3 is the only tubulin glycylase expressed in the human retina, essential for monoglycylation of microtubules of the cilium, including the retinal photoreceptor cilium, to prevent cilium degeneration and retinal degeneration. Our preliminary data suggest that rescue of tubulin glycylation through overexpression of TTLL3 is sufficient to rescue cilium number in PRPF6 and PRPF31 mutant cells, suggesting that this defect underlies the cellular defect and may represent a potential target for therapeutic intervention in this group of disorders.
cilia, ciliopathies, photoreceptor, pre-mRNA splicing, retinitis pigmentosa
1664-8021
1009430
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Villa Vasquez, Suly Saray
eb8fd041-ed3d-4f90-9094-12ab7c83163b
Lord, Jenny
e1909780-36cd-4705-b21e-4580038d4ec6
Karthik, Varshini
ed4bc3d4-1f3c-4217-b985-e523ec58ebcc
Cheung, Man Kim
8553ef1d-7690-4b23-b10a-5d89eca558c1
Lakowski, Jörn
1856e739-982a-412a-87c7-abf1610f5384
Wheway, Gabrielle
2e547e5d-b921-4243-a071-2208fd4cc090
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Villa Vasquez, Suly Saray
eb8fd041-ed3d-4f90-9094-12ab7c83163b
Lord, Jenny
e1909780-36cd-4705-b21e-4580038d4ec6
Karthik, Varshini
ed4bc3d4-1f3c-4217-b985-e523ec58ebcc
Cheung, Man Kim
8553ef1d-7690-4b23-b10a-5d89eca558c1
Lakowski, Jörn
1856e739-982a-412a-87c7-abf1610f5384
Wheway, Gabrielle
2e547e5d-b921-4243-a071-2208fd4cc090

Nazlamova, Liliya, Villa Vasquez, Suly Saray, Lord, Jenny, Karthik, Varshini, Cheung, Man Kim, Lakowski, Jörn and Wheway, Gabrielle (2022) Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations. Frontiers in Genetics, 13, 1009430, [1009430]. (doi:10.3389/fgene.2022.1009430).

Record type: Article

Abstract

Retinitis pigmentosa (RP) is the most common cause of hereditary blindness, and may occur in isolation as a non-syndromic condition or alongside other features in a syndromic presentation. Biallelic or monoallelic mutations in one of eight genes encoding pre-mRNA splicing factors are associated with non-syndromic RP. The molecular mechanism of disease remains incompletely understood, limiting opportunities for targeted treatment. Here we use CRISPR and base edited PRPF6 and PRPF31 mutant cell lines, and publicly-available data from human PRPF31+/− patient derived retinal organoids and PRPF31 siRNA-treated organotypic retinal cultures to confirm an enrichment of differential splicing of microtubule, centrosomal, cilium and DNA damage response pathway genes in these cells. We show that genes with microtubule/centrosome/centriole/cilium gene ontology terms are enriched for weak 3′ and 5′ splice sites, and that subtle defects in spliceosome activity predominantly affect efficiency of splicing of these exons. We suggest that the primary defect in PRPF6 or PRPF31 mutant cells is microtubule and centrosomal defects, leading to defects in cilium and mitotic spindle stability, with the latter leading to DNA damage, triggering differential splicing of DNA damage response genes to activate this pathway. Finally, we expand understanding of “splicing factor RP” by investigating the function of TTLL3, one of the most statistically differentially expressed genes in PRPF6 and PRPF31 mutant cells. We identify that TTLL3 is the only tubulin glycylase expressed in the human retina, essential for monoglycylation of microtubules of the cilium, including the retinal photoreceptor cilium, to prevent cilium degeneration and retinal degeneration. Our preliminary data suggest that rescue of tubulin glycylation through overexpression of TTLL3 is sufficient to rescue cilium number in PRPF6 and PRPF31 mutant cells, suggesting that this defect underlies the cellular defect and may represent a potential target for therapeutic intervention in this group of disorders.

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Accepted/In Press date: 19 August 2022
Published date: 13 September 2022
Additional Information: Copyright © 2022 Nazlamova, Villa Vasquez, Lord, Karthik, Cheung, Lakowski and Wheway.
Keywords: cilia, ciliopathies, photoreceptor, pre-mRNA splicing, retinitis pigmentosa

Identifiers

Local EPrints ID: 471594
URI: http://eprints.soton.ac.uk/id/eprint/471594
ISSN: 1664-8021
PURE UUID: d38c05c0-dfc0-4357-ad2c-5c31c0fc603a
ORCID for Suly Saray Villa Vasquez: ORCID iD orcid.org/0000-0002-6317-752X
ORCID for Jenny Lord: ORCID iD orcid.org/0000-0002-0539-9343
ORCID for Jörn Lakowski: ORCID iD orcid.org/0000-0003-4214-7580
ORCID for Gabrielle Wheway: ORCID iD orcid.org/0000-0002-0494-0783

Catalogue record

Date deposited: 14 Nov 2022 17:49
Last modified: 18 Mar 2024 03:58

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Contributors

Author: Liliya Nazlamova
Author: Suly Saray Villa Vasquez ORCID iD
Author: Jenny Lord ORCID iD
Author: Varshini Karthik
Author: Man Kim Cheung
Author: Jörn Lakowski ORCID iD

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