Indiana University-Purdue University Indianapolis (IUPUI) / During RNA Polymerase II (RNAPII) transcription, a dynamic network of
protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and
termination. Taking a proteomics approach to study RNAPII transcription can offer a
comprehensive view of the regulatory mechanisms mediated by PPIs within the
transcription complex. However, traditional affinity purification mass spectrometry (APMS)
methods have struggled to quantitatively capture many of the more dynamic, less
abundant interactions within the elaborate RNAPII transcription interactome. To combat
this challenge, we have developed and optimized a quantitative AP-MS based
method termed Disruption-Compensation (DisCo) Network Analysis that we coupled
with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to
investigate the PPIs that regulate RNAPII transcription.
In the first study, TMT-DisCo network analysis was used to analyze how
perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5
(DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network.
TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight
into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII
elongation complex. The observed changes in the RNAPII interactome also reveal the
distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo
provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate
complex processes.
The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and
Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to
proceed through elongation, resulting in more arrested RNAPII that requires proteasomal
degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/
recycling and toward de novo expression and newly assembled RNAPII, aided
by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the
26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a
model of the nuclear proteasome functioning as a chaperone during transcription
initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex
with a hyperphosphorylated RNAPII CTD. / 2024-08-16
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/29836 |
| Date | 08 1900 |
| Creators | Burriss, Katlyn Hughes |
| Contributors | Mosley, Amber L., Georgiadis, Millie M., Goebl, Mark G., Turchi, John J. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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