Pre-mRNA splicing is carried out by the spliceosome, a dynamic molecular machine composed of five small nuclear RNAs (snRNAs) bound to proteins (snRNPs), and other protein complexes. The spliceosome undergoes a multi-step cycle of assembly, activation, catalysis, and disassembly during each splicing event. The U6 snRNA is important for coordinating metal ions within the spliceosome catalytic core that are required for transesterification reactions in splicing. Furthermore, the U6 snRNP must undergo a dynamic conformational change during spliceosome assembly, as the highly structured U6 snRNA must transition from its conformation within the U6 snRNP into a complex that is paired with U4 snRNA, the U4/U6 di-snRNP. Mutations in the U6 snRNA-binding region of U4 snRNA (RNU4-2) have been linked to neurodevelopmental disorders (Greene et al. 2024; Chen et al. 2024). To date, the only known U6 snRNP structure is from S. cerevisiae (Montemayor et al. 2018), which is different from other metazoan U6 snRNPs and lacks the N-terminal half-a-tetratricopeptide (HAT) domain commonly found in Prp24 proteins from other organisms, including humans. HAT domains are thought to be dimerization domains. We have studied the structure of the U6 snRNP from S. pombe , which shares a high degree of sequence similarity with the human U6 snRNP. We used CRISPR to engineer an endogenous TAP-tag on Prp24 for purification and compositional analysis of associated RNA and proteins using biochemical methods, mass spectrometry, and negative stain electron microscopy. These data show that the U6 snRNP is a dimeric 450 kDa complex, comprised of U6 snRNA, Prp24, and the Lsm2-8 complex. We further show the U6 snRNP is capable of forming a stable complex with U4 RNA in vitro. Preliminary EM and its biochemical characterization data for this complex will be presented.
Kume et al. (Sun,) studied this question.