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.
Highlights
Next-generation sequencing studies of cancer cells have identified mutations affecting important protein domains in RNA processing factors involved in 30 splice site (30 ss) selection, first described in myelodysplasias [1]
We report residues required for correct folding/stability of each protein and map functional RNA recognition motifs (RRMs) substitutions on to existing high-resolution structures of U2AF65 and PUF60
We have found that RRM substitutions can cause splicing defects of their target 30 ss or their own transcripts, which may further enhance the mRNA diversity of cancer cells
Summary
Next-generation sequencing studies of cancer cells have identified mutations affecting important protein domains in RNA processing factors involved in 30 splice site (30 ss) selection, first described in myelodysplasias [1]. U2AF1 (U2AF35), the mutations are restricted to missense variants that encode just a few amino acids [1,2,3]. These alterations can directly impair contacts with precursor messenger RNAs (pre-mRNAs), as exemplified by substitutions in U2AF35 zinc-finger domains [4], or may act indirectly. Cancer-associated mutations in the HEAT domains of SF3B1 [1] were proposed to lead to changes in the positioning of other RNA-binding proteins, including the 65 kilodalton (kD) subunit of the. Apart from SF3B1 and U2AF1, clonal mutations have been found in genes encoding other 30 ss recognition factors, including U2AF2 and PUF60, but at lower frequencies [1,9].
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