GPATCH11 ortholog Sap34 regulates pre-mRNA splicing by interacting with early spliceosomal complexes in...

Pre-mRNA splicing is an essential step in gene expression regulation. It is mediated by the spliceosome, a large ribonucleoprotein complex that undergoes dynamic structural and compositional rearrangements during each splicing cycle. Although the mechanisms of splicing and the roles of main spliceosomal components are well defined, the identities and functions of transiently associated spliceosomal proteins remain incompletely understood. Here, we investigated the molecular function of the poorly characterized G-patch domain-containing protein SPAC6F6.19 (herein Sap34, for spliceosome-associated protein of 34 kDa) in Schizosaccharomyces pombe, an ortholog of human GPATCH11. Using affinity purification and a yeast two-hybrid assay, we analyzed its interactome and identified its interaction partners. In addition, long-read sequencing was employed to assess Sap34-dependent changes in splicing efficiency. We found that Sap34 forms a complex with components of the U2 small nuclear ribonucleoprotein (snRNP) and the U4/U6 × U5 tri-snRNP, which are required for early spliceosome assembly and activation. Furthermore, we defined the interaction specificity of Sap34 with splicing proteins, demonstrating the importance of its C-terminal region for binding to Sap61 and Ini1, and of its G-patch domain for interaction with ATP-dependent RNA helicase Prp43, suggesting that G-patch domain of Sap34 may contribute to the regulation of Prp43 activity in early spliceosomes. Notably, we showed that deletion of sap34 leads to a global reduction in splicing efficiency, predominantly associated with increased intron retention. Together, these findings identify Sap34 as a previously unrecognized and important G-patch domain-containing protein regulating the early steps of pre-mRNA splicing in fission yeast.