Key result
SF3B inhibitors did not alter U2 snRNA secondary structure compared to A-complex, indicating that splicing complex instability stems from disrupted protein interactions.
Why the study?
SF3B inhibited-complex is less stable than A-complex, raising the hypothesis that SF3B inhibitors interfere with U2 snRNA secondary structure changes needed for branch helix formation.
SF3B inhibitors do not alter U2 snRNA secondary structure, suggesting they interfere with protein interactions rather than RNA conformation, while SF3A/B proteins influence the structure of Stem I.
Refines SF3B inhibitor mechanisms toward protein interactions; leaves open translation to cardiovascular splicing therapies.
U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.
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Urabe et al. (2021) studied this question. SF3B inhibitors vs. Uninhibited A-complex was evaluated on U2 snRNA secondary structure conformation. SF3B inhibitors did not alter U2 snRNA secondary structure compared to A-complex, indicating that splicing complex instability stems from disrupted protein interactions.
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