Investigations of how phosphorylation on histone H3T45 orchestrates the RNA Exosome complex to mediate RNA processing Restricted; Files Only
Zhang, Maggie (Spring 2024)
Abstract
Histone proteins undergo extensive modifications, and these post-translational modifications (PTMs) play pivotal roles in regulating nucleosome dynamics and functions and facilitate the recruitment of effector proteins, essential for executing various chromatin-related functions. In this study, our focus was directed towards elucidating the mechanistic underpinnings of a novel histone post-translational modification (PTM), phosphorylation on histone H3.3 Thr45 (pH3T45), which is located at the DNA entry/exit site of a nucleosome. Upon the preliminary finding that H3 peptide carrying pH3T45 can recruit several subunits of the RNA exosome complex, this study aims to understand how pH3T45 orchestrates the RNA exosome complex to mediate co-transcriptional RNA processing in the context of events that compromise genome stability, such as RNA polymerase II backtracking and DNA-RNA hybrids, or R-loops, formation. Genetic interaction studies conducting in S. cerevisiae model system between phosphorylation-null mutation H3T45A and different types of R-loop accumulating mutation suggested that pH3T45 may be indirectly related to R-loops or related to a specific category or R-loops originating from defects in co-transcriptional RNA processing. We were unable to validate the interaction between pH3T45 and the RNA exosome complex in the co-immunoprecipitation performed in engineered T47D human breast cancer cells which carry wildtype H3.3, phosphorylation-null mutation H3.3T45A, and phosphomimic mutation H3.3T45E. Based on the nature of pH3T45 and the potential interacting mode between pH3T45 and the RNA exosome complex, improvements in future experimental design were proposed. Overall, this study has established a foundational framework and highlighted areas for refinement in future mechanistic investigations regarding how the novel histone post-translational modification pH3T45 orchestrates the RNA exosome complex to regulate co-transcriptional RNA processing events, with particular attention to R-loops and RNAPII backtracking.
Table of Contents
1. Introduction
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1.1 Nucleosomes, Histones, and Post-translational Modifications (PTMs)
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1.2 Phosphorylation on Histone H3 Threonine 45 and PI3K Signaling
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1.3 Preliminary Investigation to Define the Regulatory Networks of pH3T45
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1.4 The RNA Exosome Complex, RNAPII backtracking, and R-loops
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2. Materials and Methods
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3. Results
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3.1 Potential genetic interaction was observed between genes that are involved in R-loops metabolism and H3T45A.
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3.2 Co-immunoprecipitation (Co-IP) provides insights that can guide improvements in future experimental directions.
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4. Discussion
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5. Supplemental Materials
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6. References
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File download under embargo until 24 May 2026 | 2024-04-14 17:57:53 -0400 | File download under embargo until 24 May 2026 |
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