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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

Efficient co-transcriptional splicing is thought to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. In this work, it is demonstrated that inhibition of the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells caused widespread intron retention and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these events, suggesting that unspliced introns by themselves do not cause excessive R- loops. R-loop gains were instead driven by readthrough transcription at a subset of stress-response genes, defining a new class of aberrant “downstream of genes” (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss resulting from accumulation of promoter-proximal paused RNA polymerases and defective elongation associated with premature termination. Thus, SF3B1- targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in the global R-loop landscape.

    Cover page: SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2021)
    R-loops are non-B DNA structures that form during transcription when the nascent RNA anneals to the template DNA strand forming a RNA:DNA hybrid. Understanding the genomic distribution and function of R-loops is an important goal, since R-loops have been implicated in a number of adaptive and maladaptive processes under physiological and pathological conditions. Based on R-loop mapping datasets, we propose the existence of two main classes of R-loops, each associated with unique characteristics. Promoter-paused R-loops (Class I) are short R-loops that form at high frequency during promoter-proximal pausing by RNA polymerase II. Elongation-associated R-loops (Class II) are long structures that occur throughout gene bodies at modest frequencies. We further discuss the relationships between each R-loop class with instances of genome instability and suggest that increased class I R-loops, resulting from enhanced promoter-proximal pausing, represent the main culprits for R-loop mediated genome instability under pathological conditions.
      Cover page: Defining R-loop classes and their contributions to genome instability
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2021)
      R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological favorability. In recent years, R-loops have been implicated in a variety of adaptive and maladaptive roles and have been linked to genomic instability in the context of human disorders. As a consequence, the accurate mapping of these structures in genomes is of high interest to many investigators. DRIP-seq (DNA:RNA Immunoprecipitation followed by high throughput sequencing) is described here. It is a robust and reproducible technique that permits accurate and semi-quantitative mapping of R-loops. A recent iteration of the method is also described in which fragmentation is accomplished using sonication (sDRIP-seq), which allows strand-specific and high-resolution mapping of R-loops. sDRIP-seq thus addresses some of the common limitations of the DRIP-seq method in terms of resolution and strandedness, making it a method of choice for R-loop mapping.
        Cover page: Mapping R-Loops and RNA:DNA Hybrids with S9.6-Based Immunoprecipitation Methods.
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