Abstract
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.
Highlights
The U2 small nuclear ribonucleoprotein particle is a vital component of the human spliceosome, which is the macromolecular complex responsible for pre-mRNA splicing during eukaryotic gene expression
The unchanged chemical probing reactivities and protections within the different regions of the U2 snRNA show that the SF3B inhibitors have little impact on U2 snRNA structure in both the spliceosome and in the small nuclear ribonucleoprotein particle (snRNP) under the conditions that we tested. Both functional A-complex and SI-complex spliceosomes have reactivity patterns that indicate the presence of the upper region of Stem I, the branch helix and Stem IIa and IIb. This means that the BSL unwound, and that U2 snRNA BPRS engaged the intron in the branch helix before the SF3B inhibitor spliceostatin A (SSA) impacted assembly
In the A-complex structure, the branch helix is held in place by SF3B1 in a closed conformation, in which the branch point adenosine is sequestered in a pocket formed between two SF3B1 HEAT repeats [8]
Summary
The U2 small nuclear ribonucleoprotein particle (snRNP) is a vital component of the human spliceosome, which is the macromolecular complex responsible for pre-mRNA splicing during eukaryotic gene expression. U2 snRNP is composed of the U2 snRNA, core proteins, and SF3A and SF3B subcomplexes [1, 2].
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