Precursor RNA Properties Driving Cryptic 3’ Splice Site Selection in SF3B1 Mutant Malignancies

Author

• Austin A. Herbert, Clemson UniversityFollow

Date of Award

5-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics and Biochemistry

Committee Chair/Advisor

Lela Lackey

Committee Member

Trudy Mackay

Committee Member

Rich Steet

Committee Member

Liangjiang Wang

Abstract

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3’ splice site selection. SF3B1 is commonly mutated in myelodysplastic syndromes and other blood cancers with the K700E mutation being the most frequent. This mutation of SF3B1 induces cryptic 3’ splice site activation in hundreds of genes. Introduction of such cryptic 3’ splice sites can shift the reading frame of protein coding transcripts, causing the transcript to be tagged for nonsense-mediated decay or resulting in the production of a truncated protein. Both these scenarios can lead to down-regulated protein expression. Sequence and structural properties of precursor RNAs with cryptic 3’ splice sites specifically activated in SF3B1 mutants are poorly understood. The overarching goal of my thesis research has been to investigate the intrinsic sequence properties and secondary structural characteristics of transcripts mis-spliced by SF3B1 K700E mutant spliceosomes. Chapter One introduces potential mechanisms contributing to cryptic 3’ splice site activation by SF3B1 mutants. Chapter Two reviews the current understanding of how RNA structure influences gene expression, including an overview of methods used in this thesis research. Chapter Three reports that SF3B1 mutation-sensitive cryptic 3’ splice site are flanked on both sides by polypyrimidine tracts and that their paired canonical 3’ splice sites are significantly weaker than 3’ splice sites unaffected by SF3B1 mutation. Additionally, the intronic sequences upstream of SF3B1 mutation-sensitive cryptic 3’ splice sites are found to be much more flexible than those resistant to SF3B1 mutation. Finally, in Chapter Four, we utilize a synthetic MAP3K7 intron, which is sensitive to SF3B1 K700E mutation, to interrogate the effect of hundreds of different mutants on structural changes and splicing efficiency. We observed a significant increase in cryptic 3’ splice site use when knocking out candidate branch point and RNA binding protein motifs. Altogether, dissertation delineates the sequence and structural properties of SF3B1-sensitive precursor RNA 3’ splice junctions, leading to framework for development of SF3B1 mutation-specific therapeutics.

Recommended Citation

Herbert, Austin A., "Precursor RNA Properties Driving Cryptic 3’ Splice Site Selection in SF3B1 Mutant Malignancies" (2026). All Dissertations. 4243.
https://open.clemson.edu/all_dissertations/4243

Author ORCID Identifier

https://orcid.org/0000-0002-6283-7784