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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2024)

RNA editing enzymes, such as those in the APOBEC and ADAR families, modify RNA transcripts with nucleotide-level specificity, playing critical roles in cellular differentiation and function. Beyond their endogenous roles, these enzymes have been repurposed as tools for identifying RNA-binding protein (RBP) binding sites when fused to RBPs of interest. However, the lack of efficient computational methods to identify editing events and distinguish them from off-target effects, genetic variations, and sequencing errors has posed a significant challenge. In the first chapter of this thesis, we introduce FLARE, a Snakemake-based computational pipeline for analyzing RNA editing enrichment. FLARE identifies statistically enriched regions for various types of RNA editing, including cytosine-to-uracil (C-to-U) and adenosine-to-inosine (A-to-I) conversions. Applied to C-to-U data from an RBFOX2-APOBEC1 STAMP experiment, FLARE demonstrates high specificity in detecting RBFOX2 binding sites. Additionally, FLARE effectively identifies regions of endogenous A-to-I hyperediting, underscoring its versatility. In the second chapter, we present MARINE (Multi-Core Algorithm for Rapid Identification of Nucleotide Edits), a fast and efficient Python-based tool for analyzing RNA

editing across diverse sequencing data types. MARINE is optimized for single-cell resolution, enabling more detailed, cell type-specific mapping of the RNA editing landscape. Using MARINE to analyze an immune cell dataset, we uncover a potential role for RNA editing in the proliferation and differentiation of B cells. I also demonstrate MARINE's utility in assessing RBFOX2-APOBEC1 fusion-directed editing in single cells, in the context of the STAMP technology developed by the Yeo lab. MARINE empowers granular investigations into transcriptomic diversity, enabling insights into the impact of RNA editing in both health and disease contexts.

    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    The development and functional potential of metazoan cells is dependent on combinatorial roles of transcriptional enhancers and promoters. Macrophages provide exceptionally powerful model systems for investigation of mechanisms underlying the activation of cell-specific enhancers that drive transitions in cell fate and cell state. Here, we review recent advances that have expanded appreciation of the diversity of macrophage phenotypes in health and disease, emphasizing studies of liver, adipose tissue, and brain macrophages as paradigms for other tissue macrophages and cell types. Studies of normal tissue-resident macrophages and macrophages associated with cirrhosis, obese adipose tissue, and neurodegenerative disease illustrate the major roles of tissue environment in remodeling enhancer landscapes to specify the development and functions of distinct macrophage phenotypes. We discuss the utility of quantitative analysis of environment-dependent changes in enhancer activity states as an approach to discovery of regulatory transcription factors and upstream signaling pathways.
      Cover page: Exploiting dynamic enhancer landscapes to decode macrophage and microglia phenotypes in health and disease
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2024)
      RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.
        Cover page: High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissuesCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2024)
        The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1β complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1β coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1β complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.
          Cover page: A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activationCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2024)
          RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.
            Cover page: Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studiesCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2023)
            The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1β with the NCoR/HDAC3 complex, resulting in the activation of PGC1β and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.
              Cover page: RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1β- and RNA-dependent co-activator of osteoclast gene expressionCreative Commons 'BY' version 4.0 license
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