Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155 V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1mutant disease phenotypes may have a complex origin on the spliceosome.

In eukaryotes, a mRNA maturation step called splicing removes non‐coding intronic regions from pre‐mRNAs. Splicing is conducted by an enzyme called the spliceosome which uses a variety of protein and snRNA components in order to perform its catalytic function. One of these highly conserved components is the U2 snRNP protein SF3B1. In humans, mutation of SF3B1 can lead to improper regulation of alternative splicing. Many human diseases such as myelodysplastic syndromes (MDS), endometrial cancer (UCEC), bladder carcinoma (BLCA), and acute myeloid leukemia (LAML) have been linked to SF3B1 mutation and dysregulation of alternative splicing.The goal of this study is to introduce SF3B1 mutations observed in UCEC, BLCA, or LAML cancer patients into the homologous yeast protein (HSH155) to gain further insight into the consequences of these mutations on splicing. I have used site‐directed mutagenesis to incorporate these mutations into the HSH155 gene on plasmids and then shuffled these plasmids into a budding yeast strain in which the HSH155 gene was deleted from the genome. I then used temperature‐dependent growth assays and an in vivo splicing reporter to measure phenotypic responses due to these point mutations. These experiments will identify how cancer‐linked mutations change conserved, fundamental steps carried out by the spliceosome and how perturbation of these steps can led to changes in alternative splicing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AbstractMutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.