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Open Access Publications from the University of California

Scholarly Works (32 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2022)
APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare mutations by micro-partitioning bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events in cell lines or patient samples. For complete details on the use and execution of this protocol, please refer to Jalili et al. (2020) and Oh et al. (2021).
    Cover page: A digital PCR-based protocol to detect and quantify RNA editing events at hotspots
    • Thesis
    • Peer Reviewed
    UC Irvine Electronic Theses and Dissertations (2024)

    The innate immune system is the first line of defense, providing a rapid, broad-spectrum defense during viral infections. Innate immunity relies on pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs), the unique molecular patterns found in pathogens, and mount appropriate downstream immune responses. There are multiple PRRs, which allow cells to recognize a wide range of viruses. Upon activation, PRRs initiate a complex cascade of signaling pathways involving numerous adaptor proteins, kinases, and transcription factors to orchestrate the expression of inflammatory genes encoding cytokines, chemokines, and other anti-viral immune proteins important in inhibiting viral replication and propagation. Meanwhile, viruses continuously evolve strategies to evade the host’s innate immune detection, bringing additional pressure to develop multiple layers of protective mechanisms. Moreover, the innate immune system must be tightly regulated to prevent its aberrant activation by distinguishing between self and non-self molecules. Given the complexity of the innate immune system, it is essential to thoroughly investigate the roles and regulatory mechanisms of immune factors. Therefore, the aim of this dissertation is to elucidate novel regulators and their functions within various pathways of innate immunity.

    Chapter 1 focuses on identifying a novel regulator in the OAS-RNase L pathway. OAS-RNase L is an important antiviral pathway triggered in response to diverse viral infections. Upon double-stranded RNA binding, OAS enzymes synthesize 2’-5’ linked oligoadenylates (2-5A), leading to the activation of latent endonuclease RNase L. Once activated, RNase L leads to rapid decay of host and viral RNAs and shut down host translation machinery. As many viruses have developed strategies to counteract different steps of the OAS/RNase L pathway, it is crucial to fully characterize the regulators involved in this signaling cascade to understand how viruses evade our innate immunity. To this date, whether there are additional factors other than OAS3 and RNase L is not well known. I developed CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identified IRF2 as a key regulator of OAS3. I demonstrated that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 cooperates with the interferon response through STAT2 to further enhance OAS3 expression after viral infection. I propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense and giving host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

    Chapter 2 demonstrates a novel function of APOBEC3B (A3B) in protecting the assemblies of stress granules (SGs) during viral infections. A3B belongs to the family of cytosine deaminase APOBEC3 (A3) family that counteracts viruses, primarily by inducing mutations in the viral genomes. Interestingly, A3s have been shown to restrict viruses independently of their genome editing role. However, the different mechanisms of how A3 members can suppress viral replication without deaminase activity are poorly understood and need further investigation to fill the gap in knowledge on how innate immunity protects cells against viruses. During viral infection, the activation of RNase L is crucial for limiting viral replications by causing widespread RNA decay and shutting down host translation machinery, but this activity of RNase L simultaneously results in SGs disassembly, which appears to be counterproductive for cells. In this study, I demonstrated that A3B is recruited to SGs to counteract RNase L activity and protects the mRNAs associated with SGs from RNase L-induced RNA cleavage during viral infection. This study reveals a novel deaminase-independent function of A3B as a modulator of SGs structures in response to viral infections.

    Chapter 3 identifies the regulation of APOBEC3A (A3A) expression during viral infection and cancer mutagenesis. Besides mutating viral genome to inhibit viral replication, A3A is capable of deaminating the human genome and has been shown to be one of the major causes of APOBEC mutational signatures found in patient’s tumors. However, the mechanisms by which viral infection and tumors trigger A3A expression are still poorly understood. In this study, I find that both viral infection and genotoxic stress transiently up-regulate A3A and pro-inflammatory genes using two distinct mechanisms. STAT2-mediated interferon pathway promotes A3A expression in response to foreign nucleic acids through RIG-I/MAVS signaling cascade while DNA damage and replications stress independently activate the NF-κB pathway to induce expression of A3A and other innate immune genes. This study reveals different types of stressors that lead to the expression of A3A and the mechanisms of regulations during viral infections and cancer mutagenesis.

      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2012)
      PALB2 is essential for BRCA2 anchorage to nuclear structures and for homologous recombinational repair of DNA double-strand breaks. Here, we report that the N-terminal coiled-coil motif of PALB2 regulates its self-association and homologous recombination. Monomeric PALB2 shows higher efficiency to bind DNA and promotes RAD51 filament formation with or without the inhibitory effect of Replication Protein A. Moreover, overexpression of the PALB2 coiled-coil domain severely affects RAD51 loading to DNA damage sites suggesting a competition between PALB2 self-interaction and PALB2-BRCA1 interaction. In the presence of DNA damage, the switch between PALB2-PALB2 and PALB2-BRCA1 interactions allows the activation of HR. Controlling HR via PALB2 self-interactions could be important to prevent aberrant recombination in normal conditions and activate DNA repair when required.
        Cover page: PALB2 self-interaction controls homologous recombination
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2015)
        The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.
          Cover page: Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2018)
          The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.
            Cover page: A mitosis-specific and R loop–driven ATR pathway promotes faithful chromosome segregation
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2017)
            The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like APOBEC3A and APOBEC3B have emerged as key mutation drivers in cancer. Here, we show that APOBEC3A and APOBEC3B activities impose a unique type of replication stress by inducing abasic sites at replication forks. In contrast to cells under other types of replication stress, APOBEC3A-expressing cells were selectively sensitive to ATR inhibitors (ATRi), but not to a variety of DNA replication inhibitors and DNA-damaging drugs. In proliferating cells, APOBEC3A modestly elicited ATR but not ATM. ATR inhibition in APOBEC3A-expressing cells resulted in a surge of abasic sites at replication forks, revealing an ATR-mediated negative feedback loop during replication. The surge of abasic sites upon ATR inhibition associated with increased accumulation of single-stranded DNA, a substrate of APOBEC3A, triggering an APOBEC3A-driven feed-forward loop that ultimately drove cells into replication catastrophe. In a panel of cancer cell lines, ATRi selectively induced replication catastrophe in those harboring high APOBEC3A and/or APOBEC3B activities, showing that APOBEC3A and APOBEC3B activities conferred susceptibility to ATRi. Our results define an APOBEC-driven replication stress in cancer cells that may offer an opportunity for ATR-targeted therapy. Cancer Res; 77(17); 4567-78. ©2017 AACR.
              Cover page: APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2023)
              APOBEC3A is an antiviral DNA deaminase often induced by virus infection. APOBEC3A is also a source of cancer mutation in viral and nonviral tumor types. It is therefore critical to identify factors responsible for APOBEC3A upregulation. Here, we test the hypothesis that leaked mitochondrial (mt) double-stranded (ds)RNA is recognized as foreign nucleic acid, which triggers innate immune signaling, APOBEC3A upregulation, and DNA damage. Knockdown of an enzyme responsible for degrading mtdsRNA, the exoribonuclease polynucleotide phosphorylase, results in mtdsRNA leakage into the cytosol and induction of APOBEC3A expression. APOBEC3A upregulation by cytoplasmic mtdsRNA requires RIG-I, MAVS, and STAT2 and is likely part of a broader type I interferon response. Importantly, although mtdsRNA-induced APOBEC3A appears cytoplasmic by subcellular fractionation experiments, its induction triggers an overt DNA damage response characterized by elevated nuclear γ-H2AX staining. Thus, mtdsRNA dysregulation may induce APOBEC3A and contribute to observed genomic instability and mutation signatures in cancer.
                Cover page: Mitochondrial double-stranded RNA triggers induction of the antiviral DNA deaminase APOBEC3A and nuclear DNA damage.
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2013)
                Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.
                  Cover page: GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2024)
                  OAS-RNase L is a double-stranded RNA-induced antiviral pathway triggered in response to diverse viral infections. Upon activation, OAS-RNase L suppresses virus replication by promoting the decay of host and viral RNAs and inducing translational shutdown. However, whether OASs and RNase L are the only factors involved in this pathway remains unclear. Here, we develop CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identifies IRF2 as a key regulator of OAS3. Mechanistically, we demonstrate that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 works in concert with the interferon response through STAT2 to further enhance OAS3 expression. We propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense. This rapid response provides host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.
                    Cover page: A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2014)
                    One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3'-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.
                      Cover page: Breast Cancer Proteins PALB2 and BRCA2 Stimulate Polymerase η in Recombination-Associated DNA Synthesis at Blocked Replication Forks
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