Titration of SF3B1 Activity Reveals Distinct Effects on the Transcriptome and Cell Physiology
Karen S Kim Guisbert1, Isiah Mossiah1, Eric Guisbert1
1Department of Biomedical and Chemical Engineering and Sciences, Florida Institute of Technology, Melbourne, FL 32937, USA.
Abstract:
SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.
Insights
The spliceosome component SF3B1, when modulated by pladienolide B (PB), impacts gene expression and cellular physiology. Even low doses reveal distinct gene expression changes, highlighting SF3B1
Area of Science:
- Molecular Biology
- Cancer Biology
- Genetics
Background:
- SF3B1 is a crucial U2 spliceosome component frequently altered in cancer.
- Previous studies linked SF3B1 activity modulation to heat shock response (HSR) pathways.
Purpose of the Study:
- To identify genes sensitive to varying SF3B1 activity levels.
- To investigate SF3B1's broader influence on gene expression beyond splicing.
Main Methods:
- RNA sequencing was employed to analyze gene expression changes at 5 nM and 100 nM pladienolide B (PB).
- Independent reporter constructs were used to validate specific pathway alterations.
Main Results:
- Significant alterations in mRNA splicing and gene expression were observed at both low and high PB concentrations.
- SF3B1 influences gene expression independently of alternative splicing.
- Gene expression changes at low PB did not predict those at high PB, indicating differential sensitivity.
- Nonsense-mediated decay and protein-folding homeostasis pathways showed differential sensitivity to PB.
- Low PB exposure enhanced cellular protein-folding capacity.
Conclusions:
- The transcriptome exhibits high sensitivity to SF3B1 activity levels.
- SF3B1 activity is finely tuned to orchestrate mRNA splicing, gene expression, and cellular physiology.
