Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3 ' Splice-Site Selection

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Publikace nespadá pod Pedagogickou fakultu, ale pod Středoevropský technologický institut. Oficiální stránka publikace je na webu muni.cz.
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KRALOVICOVA J. BOROVSKA I. KUBÍČKOVÁ Monika LUKAVSKY Peter VORECHOVSKY I.

Rok publikování 2020
Druh Článek v odborném periodiku
Časopis / Zdroj Cancers
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
www https://www.mdpi.com/2072-6694/12/7/1865
Doi http://dx.doi.org/10.3390/cancers12071865
Klíčová slova Functional genomics; pre-mRNA splicing; 3 ' splice site; mRNA; lariat branch point; PUF60; U2AF2; gel shift assay; differential scanning fluorimetry; cancer; leukemia; driver mutation; exon inclusion
Popis 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 internalU2AF2(similar to 9%, 2/23) orPUF60(similar to 8%, 1/13) exons, indicating that cancer-associated RRM mutations can have bothcis- andtrans-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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