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Cancer-associated substitutions in RNA recognition motifs of PUF60 and U2AF65 reveal residues required for correct folding and 3’ splice-site selection

Cancer-associated substitutions in RNA recognition motifs of PUF60 and U2AF65 reveal residues required for correct folding and 3’ splice-site selection
Cancer-associated substitutions in RNA recognition motifs of PUF60 and U2AF65 reveal residues required for correct folding and 3’ splice-site selection
U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.
3′ splice site, Cancer, Differential scanning fluorimetry, Driver mutation, Exon inclusion, Functional genomics, Gel shift assay, Lariat branch point, Leukemia, MRNA, PUF60, Pre-mRNA splicing, SF3B4, U2AF2
2072-6694
1-19
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Borovska, Ivana
d2d23654-81da-45a7-8a93-3fc55826e4bc
Kubickova, Monika
639fae1a-4d88-4d2f-87b1-3eeb7df60547
Lukavsky, Peter J.
9beb2aad-36c2-48b3-8797-ef33a5642006
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Borovska, Ivana
d2d23654-81da-45a7-8a93-3fc55826e4bc
Kubickova, Monika
639fae1a-4d88-4d2f-87b1-3eeb7df60547
Lukavsky, Peter J.
9beb2aad-36c2-48b3-8797-ef33a5642006
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e

Kralovicova, Jana, Borovska, Ivana, Kubickova, Monika, Lukavsky, Peter J. and Vorechovsky, Igor (2020) Cancer-associated substitutions in RNA recognition motifs of PUF60 and U2AF65 reveal residues required for correct folding and 3’ splice-site selection. Cancers, 12 (7), 1-19, [1865]. (doi:10.3390/cancers12071865).

Record type: Article

Abstract

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

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cancers-852124-in press - Accepted Manuscript
Available under License Creative Commons Attribution.
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More information

Submitted date: 5 July 2020
Accepted/In Press date: 7 July 2020
e-pub ahead of print date: 11 July 2020
Keywords: 3′ splice site, Cancer, Differential scanning fluorimetry, Driver mutation, Exon inclusion, Functional genomics, Gel shift assay, Lariat branch point, Leukemia, MRNA, PUF60, Pre-mRNA splicing, SF3B4, U2AF2

Identifiers

Local EPrints ID: 442745
URI: http://eprints.soton.ac.uk/id/eprint/442745
ISSN: 2072-6694
PURE UUID: 15ad14a8-e73d-44dc-9c0a-78a88feadfe0

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Date deposited: 24 Jul 2020 16:45
Last modified: 25 Nov 2021 18:36

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Contributors

Author: Jana Kralovicova
Author: Ivana Borovska
Author: Monika Kubickova
Author: Peter J. Lukavsky

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