Functional and Structural Impacts of Mutations to the SF3B4:SF3B2 Interface
Creators: Poholka, Andrew N.
Publisher: name: University of Alberta Library
Year: 2024

<p>Eukaryotic genomes have a split gene structure that requires pre-mRNA to be spliced for proper gene expression. The splicing process is catalyzed by a multi-megadalton complex known as the spliceosome. This molecular machine assembles on the pre-mRNA and undergoes a series of rearrangements to catalyze a pair of transesterification reactions that excise the intron. The SF3B complex is a component of the spliceosome that plays an important role in the splicing reaction by assisting in the identification and temporary sequestration of the catalytically vital branch point adenosine. SF3B4 and SF3B2 are two components of the SF3B complex that form a specific interaction with each other and interact with the pre-mRNA upstream of the branch point adenosine. Both proteins are implicated in a variety of cancers, and haploinsufficiency of SF3B4 is associated with acrofacial dysostosis.<br>
            Although spliced introns were initially thought to be simply degraded, a growing body of literature demonstrates that introns can serve a variety of functions. Recently, two reports have shown a role for introns in the regulation of metabolism downstream of the TOR pathway, more specifically the starvation response. These studies found that in Saccharomyces cerevisiae specific introns accumulate in the stationary phase of growth. These accumulating introns sequester spliceosomes to prevent the splicing of ribosomal protein genes with introns. Removal of introns from the genome or preventing the introns from accumulating impairs the starvation response. <br>
            Interestingly, the starvation response is also inhibited by the Q24P mutation to SF3B4, which produces a phenotype that is remarkably similar to that produced by the removal of introns.  The Q24P mutation is located at the interface between SF3B4 and SF3B2, which suggests that it may disrupt the stability of the complex. This thesis aims to investigate the impact of the Q24P mutation on splicing in S. cerevisiae as well as the structural consequences of the Q24P mutation on the stability of the yeast SF3B4:SF3B2 complex alongside the homologous L28P mutation on the human SF3B4:SF3B2 complex. In vitro splicing assays showed that the MMS2 intron was spliced more efficiently in extracts derived from S. cerevisiae with the Q24P mutation in the stationary phase growing in minimal media. The ACT1 intron, on the other hand, may have been spliced less efficiently in extracts derived from S. cerevisiae with the Q24P mutation in the logarithmic and stationary phases growing in YPD media.<br>
            Structural analysis of the human SF3B4:SF3B2 complex demonstrated that the L28P mutation destabilizes the complex. The Q24P mutation causes the S. cerevisiae SF3B4:SF3B2 complex to undergo degradation, likely due to this destabilization. The impact of the Q24P mutation is dependent on phase and growth conditions, suggesting that there may be another protein factor that is mediating the effect. A literature and database search revealed FPR1 as a protein involved with the TOR pathway and regulation of ribosomal protein genes, as well as with reported interactions with SF3B4, making it a likely candidate for this factor.</p>