UC Santa Cruz

AbstractAngela Nicole Amorello Developing an in vitro thermal shift assay to assess target engagement of spliceosome inhibitors Successful human gene expression requires a balance of flexibility and accuracy. Human genes are transcribed as pre-mRNAs that contain coding exon sequences interspersed with non-coding intron sequences. Introns must be removed, and exons joined together to produce a functional mRNA by pre-mRNA splicing. Splicing is catalyzed by a ribonucleoprotein molecular machine called the spliceosome. The spliceosome is a complex enzyme with many key players and assembles piece-by-piece on each pre-mRNA. Accurate splicing requires identification of the intron/exon boundaries by the spliceosome. The spliceosome relies on identifying introns by unique sequence signatures. Poor conservation of these sequences in human introns affords the spliceosome flexibility for alternative splicing, but also necessitates tight regulation. My research focuses on U2 snRNP and its associated protein subcomplex SF3B, which are involved in branch point sequence recognition. I speculate that branch point sequence recognition by U2 snRNP in humans is reliant on SF3B-mediated interactions. I used two approaches to interrogate SF3B-mediated interactions, 1) assessing how drug binding impacts the thermal stability SF3B protein, SF3B1 and 2) determining whether SF3B1-interacting partner, U2AF is required for intron recognition.