The adaptor protein MAVS plays an essential role in the immune system's antiviral defenses. Upon sensing viral RNA in the cytosol, RIG-I-like receptors trigger MAVS to form a signalosome with other proteins and induce interferon (IFN) expression. Recent datasets have identified three regions in the MAVS protein that might bind to RNA. However, the functions underlying these RNA-binding regions (RBRs 1, 2, 3) are still not understood. In this study, we aim to identify the differential functions of these RBRs on regulating MAVS interactions. I tested the ability of FLAG-tagged MAVS constructs with different RBR deletions to induce IFNB1 when overexpressed in MAVS knockout (KO) 293T cells. Different combinatorial deletions of these RBR regions lead to differential levels of IFN induction; deletion of RBR2 abolishes IFN expression, while constructs with additional deletions of RBRs 1 or 3 slightly restores IFN expression. We then investigated where the RBRs perform their regulatory role in the MAVS signaling pathway. We hypothesized that RBRs may be involved in MAVS-TRAF binding or MAVS ubiquitination, both of which would induce differing levels of IFN expression if disrupted. I used immunoprecipitation to find that the RBR2-deleted construct abrogated MAVS-TRAF binding, while any constructs with RBR1 deleted showed increased MAVS-TRAF binding, consistent with their restoration of IFNB1 expression. I will also use immunoprecipitation to measure the influence of RBRs on HA-tagged degradative K48 or activating ubiquitin K63. If the constructs interact with these ubiquitin mechanisms, we expect HA pulldowns from RBR1 deletions to show increased MAVS-K63 binding and/or the RBR2-deletion to increase MAVS-K48 binding. This study provides analysis of key regulatory regions that control the downstream IFN production and antiviral defense through MAVS, which could expose therapeutic targets for either treating viral infection or reducing the effects of abnormal IFN production such as in autoimmune disorders.

Skin is a densely innervated sensory organ that protects us every day from environmental trauma. As a barrier organ, skin is susceptible to frequent damage that must be promptly and properly healed to prevent infection and restore sensory function. Our lab uses adult zebrafish as a model to study skin injury and repair. Adult zebrafish skin is similar in composition to human skin and transparent, lending itself to high-resolution microscopy. Previous experiments in our lab revealed that dynamic, skin-resident immune cells known as Langerhans cells (LCs) rapidly engulf cellular and axonal debris after injury in the zebrafish skin. Calcium signaling regulates phagocytosis and cell motility in other immune cells, but the role of calcium signaling in LCs is unstudied. Through skin explant assays, various injury paradigms, and confocal fluorescence microscopy, I have established a model for monitoring calcium signaling in LCs. I found that LCs exhibit rapid, transient calcium flashes under homeostatic conditions. However, upon engulfment of large cellular debris generated by precise laser-ablation of skin cells, LCs exhibit an atypical sustained calcium signal lasting an hour on average. To test the requirement of calcium during engulfment by LCs, I treated skin with the drug Thapsigargin to perturb calcium flux. I confirmed that Thapsigargin increases intracellular calcium in LCs and keeps intracellular calcium concentrations elevated for hours after drug addition. During Thapsigargin treatment, I showed that LCs formed phagocytic cups around cellular debris but engulfed fewer laser-ablated corpses compared to controls. Thapsigargin-treated LCs also experienced normal migration to a wound site. My results indicate that calcium flux regulates LC engulfment of large debris, but not through migration. Identifying the molecular mechanisms underlying LC motility and debris removal is ultimately relevant to understanding skin repair and disease states in which the wound healing response is attenuated, such as in chronic wounds.

Bacterial CRISPR immune systems defend against foreign genetic material, such as bacteriophage viruses. CRISPR systems are classified into six types with diverse protein components and mechanisms of interference. Among these, our research investigates the function of CRISPR-Cas13 systems, which uniquely target RNA rather than DNA. To overcome immunity, bacteriophages have evolved anti-CRISPR mechanisms that are designed to inhibit specific CRISPR types, restoring infection and proliferation of the viral invader. We recently discovered a novel anti-CRISPR mechanism, in which a noncoding RNA provides inhibition of CRISPR-Cas13 function. The central questions surrounding this RNA anti-CRISPR (rnAcr) are how it associates with CRISPR-Cas13 in order to inhibit its function, as well as the boundaries of its length and anticipated mechanism of inhibition. rnAcr is predicted to have three vital stem loops, which have been experimentally deleted and structurally disrupted by performing site directed mutagenesis to mutate select regions of nucleotides in each stem’s structure. We did this in order to determine if the stem loops’ structures were necessary for rnAcr’s anti-CRISPR function. We found that these were all essential for its function, which gives rise to the hypothesis that its structure is interacting with the bacterial host’s CRISPR-Cas13 system to effectuate its inhibitory mechanism. In order to test anti-CRISPR function, we conjugated a target and nontarget plasmid, in which the target plasmid would be recognized by Cas13, and cellular RNA would be cleaved, leaving no growth if no anti-CRISPR mechanism is present. We have shown that rnAcr is sufficient for anti-CRISPR function, allowing for tolerance of these target plasmids and cellular growth. rnAcr suggests a novel anti-CRISPR mechanism, as until now, the majority of reported anti-CRISPRs have been composed of small proteins produced during phage infection, suggesting rnAcr’s significant implications when considering new players in the host-bacteriophage evolutionary competition.

Cytomegalovirus (CMV) is a nuclear-replicating DNA herpesvirus that rearranges the nucleus to form a kidney bean shape during infection. CMV-induced cellular rearrangement is disrupted by knockout of a histone variant, macroH2A1. This disruption leads to significantly decreased infectious progeny for CMV. We examined how different transcriptional profiles during CMV infection of macroH2A1 knockout cells and found several host genes were misregulated in the absence of macroH2A1. One such gene is KIF1A, a kinesin-3 motor protein found in neurons. I previously found that KIF1A is induced in primary human foreskin fibroblasts (HFFs) during infection, which is unusual since KIF1A is not normally expressed in fibroblasts. Interestingly, KIF1A is not induced during CMV infection in the macroH2A1 knockout cell line. This led me to hypothesize that macroH2A1 is required for induction of KIF1A expression. To test this hypothesis, I overexpressed KIF1A in HFFs to establish if I can rescue the defect in infectious progeny. Wild-type and macroH2A1 knockout HFFs transduced with a plasmid containing the KIF1A gene are analyzed using Western blotting and plaque assays to determine KIF1A expression and viral titers. I anticipate that the defect is rescued by overexpressing KIF1A in macroH2A1 knockout cells. CMV is the leading infectious cause of birth defects in the United States, making its mechanisms of infection a key area of study for development of antiviral therapies.

The nucleolus is an essential subnuclear organelle that performs central regulatory roles in cellular metabolism, epigenetic programming, and stress signaling. In mammals, nucleoli are disassembled and rebuilt de novo with each cell division, through an elaborate assembly mechanism that has long eluded molecular characterization. This assembly process is spatiotemporally controlled by a long noncoding RNA termed the 47S pre-ribosomal RNA (47S pre-rRNA), which initiates nucleolar assembly at the site of its transcription, and for which continued expression is required to maintain nucleolar integrity. Yet, while the 47S’ roles in nucleating and scaffolding nucleolar architecture are well established cytologically (they were first observed nearly a century ago), the structural elements on the 47S that enable these architectural functions remain unknown. I hypothesize that an RNA domain within the 47S, termed the 5´–External Transcribed Spacer (5´–ETS), harbors the long-sought structural scaffolds of the nucleolus. To test this, I am implementing a live-cell reporter assay that will monitor, in real time, if transcripts derived from the 5´–ETS drive nucleolar localization and architecture. My approach leverages recent advancements in artificial gene synthesis and live-cell RNA imaging. A novel drug-inducible promoter will enable me to temporally control expression of 5´–ETS sequence variants in live cells. I will monitor the kinetics and efficiency with which these transcripts localize into the nucleolus by two-color live cell imaging, using the newly discovered fluorescent RNA aptamer RhoBAST, and a fluorescently tagged nucleolar marker protein. To design our negative controls, I implemented a bioinformatic pipeline that generates scrambles of long, low-complexity RNA sequences—ablating primary structure but preserving dinucleotide content. This allows us to investigate whether nucleotide composition or sequence affects nucleolar formation. We anticipate that this powerful system will set the stage for detailed molecular characterization studies, revealing the long-elusive molecular interactions that control nucleolar architecture in health and disease.

Mitochondrial calcium is essential for energy metabolism and cell survival. Deranged mitochondrial calcium leads to pathological remodeling of the heart. Little is known regarding the regulation and roles of mitochondrial calcium in cardiomyocyte growth. Mitochondrial calcium uniporter (MCU) is a major channel for mitochondrial calcium uptake. Germline knockout of MCU on the inbred C57BL/6 background is lethal. However, αMHC-Cre-driven MCU deletion in the heart just before birth yields viable offspring with normal heart function. In this study, we will use human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) to study the regulation and roles of MCU in cardiomyocyte growth. First, the expression of MCU in iPSCs and iPSC-CMs at different stages of their differentiation and maturation process will be determined at mRNA and protein levels. Then, we will delete MCU gene in undifferentiated iPSCs and follow a protocol to differentiate them into beating cardiomyocytes. The iPSC-CMs will be monitored for their morphological changes, cardiac troponin T expression, and electric pacing-induced calcium transients and cell contraction. The proliferation of iPSC-CMs will also be evaluated by using BrdU staining and molecular markers. This study will demonstrate how MCU expression changes during the differentiation and maturation of iPSC-CMs and whether it plays a role in cardiomyocyte growth.