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PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM

PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM
PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM
PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3′ splice sites (3′ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3′ss and branch points of a PUF60-dependent exon and the 3′ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3′ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.
0305-1048
6166-6187
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Sevcikova, Ivana
23cc3e1e-c7fa-4a50-9314-3e7e438c8359
Stejskalova, Eva
2dbff187-cf09-43da-8374-c6e3c4022101
Obuca, Mina
422f4db0-575a-42a3-930b-4ed2c04a4533
Hiller, Michael
edf3deb9-c9dd-4a4e-ba98-f3a9d001ea88
Stanek, David
fa7f7c51-6c23-4823-8279-f87b09f4d29b
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Sevcikova, Ivana
23cc3e1e-c7fa-4a50-9314-3e7e438c8359
Stejskalova, Eva
2dbff187-cf09-43da-8374-c6e3c4022101
Obuca, Mina
422f4db0-575a-42a3-930b-4ed2c04a4533
Hiller, Michael
edf3deb9-c9dd-4a4e-ba98-f3a9d001ea88
Stanek, David
fa7f7c51-6c23-4823-8279-f87b09f4d29b
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e

Kralovicova, Jana, Sevcikova, Ivana, Stejskalova, Eva, Obuca, Mina, Hiller, Michael, Stanek, David and Vorechovsky, Igor (2018) PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM. Nucleic Acids Research, 46 (12), 6166-6187. (doi:10.1093/nar/gky389).

Record type: Article

Abstract

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3′ splice sites (3′ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3′ss and branch points of a PUF60-dependent exon and the 3′ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3′ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

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NAR 2018 - PUF60 - Accepted Manuscript
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More information

Accepted/In Press date: 1 May 2018
e-pub ahead of print date: 18 May 2018
Published date: 6 July 2018

Identifiers

Local EPrints ID: 420918
URI: http://eprints.soton.ac.uk/id/eprint/420918
ISSN: 0305-1048
PURE UUID: 35902407-14fc-49a6-aa29-6df78271193b

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Date deposited: 18 May 2018 16:30
Last modified: 25 Nov 2021 17:25

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Contributors

Author: Jana Kralovicova
Author: Ivana Sevcikova
Author: Eva Stejskalova
Author: Mina Obuca
Author: Michael Hiller
Author: David Stanek

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