Impact of cancer-associated mutations in Hsh155/SF3b1 HEAT repeats 9-12 on pre-mRNA splicing in Saccharomyces cerevisiae

Abstract

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.

Sequence Divergence and Functional Specializations of the Ancient Spliceosomal SF3b: Implications in Flexibility and Adaptations of the Multi-Protein Complex

“…Therefore, cancer-causing residue substitutions might have influence on the pre-mRNA binding in species having such substitutions. Interestingly, when cancer-causing mutations of some of these sites (Leu822, Glu862, Glu902 and Arg957) are introduced experimentally in yeast cells, they do not show any growth defect ( Kaur et al, 2020 ). Therefore, the observed substitution patterns suggest that although specific residue types at these positions are critical in human SF3b1 and their mutations may lead to cancers, we observe that they are not uniformly selected across eukaryotes.…”

Rewards of divergence in sequences, 3-D structures and dynamics of yeast and human spliceosome SF3b complexes

“…It has been shown by earlier mutational studies that BPS recognition is largely conserved between yeast and humans and proofreading of BPS selection is likely regulated in multiple stages in the spliceosome ( Carrocci et al., 2017 ; Kaur et al., 2020 ; Tang et al., 2016 ). Here, we have investigated the potential role of SF3b6, that is unique to humans and binds at the N-terminal extension of SF3b1, during pre-B to B act stages of spliceosome through various analyses such as NMA, perturbation-response scanning and structural networks.…”

Saccharomyces cerevisiaeEcm2 Modulates the Catalytic Steps of pre-mRNA Splicing

“…Xu and Friesen provided ample evidence that Ecm2 plays a role in spliceosome activation (Xu and Friesen 2001). We and others have previously noted that key players in the activation process such as the U2 snRNP protein Hsh155/SF3B1 and U2/U6 helix I exhibit genetic interactions with a cold-sensitive (cs) mutant of the DEAH-box ATPase Prp2 (Prp2 Q548N ) (Kaur et al 2020;Carrocci et al 2017;Wlodaver and Staley 2014).…”

“…Primer extension was performed with IR dye conjugated probes yAC6: /5IRD700/GGCACTCATGACCTTC and yU6: /5IRD700/GAACTGCTGATCATCTCTG. purchased from Integrated DNA Technologies (Skokie, IL USA) (Carrocci et al 2017;Kaur et al 2020). Gels were imaged with the Amersham IR Typhoon 5 (GE Healthcare) excitation at 685 nm, emission filter 720BP20, PMT voltage of 700V, and µm pixel size.…”