Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Simon, Matthew

Abstract

The dynamic life cycle of an RNA begins with transcription initiation and concludes when the mature transcript is degraded to monomer nucleotides. The early steps of RNA polymerase II (RNAPII) transcription, initiation and promoter-proximal pausing in metazoans, are the most complex and tightly orchestrated steps. To synthesize a single mature transcript,dozens of individual proteins must be assembled around a promoter to initiate transcription. In a reliable fashion, the initiated polymerase is halted only 20 to 60 base pairs downstream of the transcription start site (TSS) in a phenomenon called promoter-proximal pausing. Pausing is induced by a small number of additional factors, but is known to facilitate the assembly of important transcription elongation and RNA processing factors on RNAPII before entering productive elongation. RNAPII then synthesizes a nascent transcript that can be of more than a megabase in length. The transcript is processed, and, in most cases, the mature transcript is then exported from the nucleus to the cytoplasm where it is eventually degraded. Probing the dynamics of RNA synthesis and degradation can be challenging because standard RNA sequencing (RNA-seq) methods provide only a steady-state snapshot of gene expression. Metabolic labeling and nucleotide-recoding chemistry with RNA-seq (NR-seq) has proven to be a powerful tool to dissect the intricacies of the RNA synthesis pathway because it provides an extra temporal dimension to RNA-seq data. Here I describe my work demonstrating incremental improvements in the handling of metabolically labeled RNA and analysis of RNA-seq data containing chemically induced mutations. I show that newly synthesized RNA can be specifically lost during RNA extraction, biasing NR-seq data against mutation-containing reads. In addition, I improved data analysis by demonstrating that implementation of a three-base alignment strategy improves alignment of mutation-containing reads. Furthermore, I apply these improved protocols in several collaborative efforts using TimeLapse-seq and transient-transcriptome-TimeLapse-seq (TT-TL-seq) to characterize the dynamics of mature RNA and transcribing RNAPII. I describe the development of Start-TimeLapse-seq (STL-seq) as the first method to directly measure the kinetics of promoter-proximal pausing in a non-perturbing, genome-wide, and TSS-specific manner. I show that STL-seq reliably quantifies the turnover of short, capped RNA transcripts associated with RNAPII at the pause site and this information accurately captures the behavior of paused RNAPII. STL-seq detects changes in paused RNAPII turnover upon a perturbation of steady-state conditions and can be used to unambiguously assign these changes to effects on pause release or premature termination. This work revealed the distinct principles of regulation of release into elongation and premature termination at the promoter-proximal pause site. Moving forward, STL-seq will be a powerful tool to dissect the mechanism and regulation of promoter-proximal pausing. Finally, I describe work pursuing my proposed model for the disease mechanism of a rare genetic disorder, X-Linked Dystonia Parkinsonism (XDP). XDP is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TATA-box binding protein (TBP) associated factor 1 gene (TAF1) and I present evidence that the SVA insertion gives rise to an alternative, truncated TAF1 transcript isoform (xTAF1) which encodes an xTAF1 protein lacking a functional second bromodomain. I demonstrate that xTAF1 associates with promoters more strongly than canonical TAF1 (cTAF1) and induces a redistribution of the RNAPII promoter-proximal pause site. I propose that these two effects confer a dominant-negative phenotype that could ultimately lead to the neurodegenerative phenotypes observed in patients.

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