Browsing by Author "Mukherjee, Priyanka"

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    RNA binding by ADAR3 inhibits adenosine-to-inosine editing and promotes expression of immune response protein MAVS
    (American Society for Biochemistry and Molecular Biology, 2022) Kurup, Reshma Raghava; Oakes, Eimile K.; Manning, Aidan C.; Mukherjee, Priyanka; Vadlamani, Pranathi; Hundley, Heather A.; Medicine, School of Medicine
    Members of the ADAR family of double-stranded RNA-binding proteins regulate one of the most abundant RNA modifications in humans, the deamination of adenosine to inosine. Several transcriptome-wide studies have been carried out to identify RNA targets of the active deaminases ADAR1 and ADAR2. However, our understanding of ADAR3, the brain-specific deaminase-deficient ADAR family member, is limited to a few transcripts. In this study, we identified over 3300 transcripts bound by ADAR3 and observed that binding of ADAR3 correlated with reduced editing of over 400 sites in the glioblastoma transcriptome. We further investigated the impact of ADAR3 on gene regulation of the transcript that encodes MAVS, an essential protein in the innate immune response pathway. We observed reduced editing in the MAVS 3' UTR in cells expressing increased ADAR3 or reduced ADAR1 suggesting ADAR3 acts as a negative regulator of ADAR1-mediated editing. While neither ADAR1 knockdown or ADAR3 overexpression affected MAVS mRNA expression, we demonstrate increased ADAR3 expression resulted in upregulation of MAVS protein expression. In addition, we created a novel genetic mutant of ADAR3 that exhibited enhanced RNA binding and MAVS upregulation compared with wildtype ADAR3. Interestingly, this ADAR3 mutant no longer repressed RNA editing, suggesting ADAR3 has a unique regulatory role beyond altering editing levels. Altogether, this study provides the first global view of ADAR3-bound RNAs in glioblastoma cells and identifies both a role for ADAR3 in repressing ADAR1-mediated editing and an RNA-binding dependent function of ADAR3 in regulating MAVS expression.
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    RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes
    (Elsevier, 2021) Mukherjee, Priyanka; Kurup, Reshma Raghava; Hundley, Heather A.; Biology, School of Science
    The past decade has seen an exponential increase in the identification of individual nucleobases that undergo base conversion and/or modification in transcriptomes. While the enzymes that catalyze these types of changes have been identified, the global interactome of these modifiers is still largely unknown. Furthermore, in some instances, redundancy among a family of enzymes leads to an inability to pinpoint the protein responsible for modifying a given transcript merely from high-throughput sequencing data. This chapter focuses on a method for global identification of transcripts recognized by an RNA modification/editing enzyme via capture of the RNAs that are bound in vivo, a method referred as RNA immunoprecipitation (RIP). We provide a guide of the major issues to consider when designing a RIP experiment, a detailed experimental protocol as well as troubleshooting advice. The RIP protocol presented here can be readily applied to any organism or cell line of interest as well as both RNA modification enzymes and RNA-binding proteins (RBPs) that regulate RNA modification levels. As mentioned at the end of the protocol, the RIP assay can be coupled to high-throughput sequencing to globally identify bound targets. For more quantitative investigations, such as how binding of an RNA modification enzyme/regulator to a given target changes during development/in specific tissues or assessing how the presence or absence of RNA modification affects transcript recognition by a particular RBP (irrespective of a role for the RBP in modulating modification levels); the RIP assay should be coupled to quantitative real-time PCR (qRT-PCR).
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    To protect and modify double-stranded RNA – the critical roles of ADARs in development, immunity and oncogenesis
    (Taylor & Francis, 2021) Erdmann, Emily A.; Mahapatra, Ananya; Mukherjee, Priyanka; Yang, Boyoon; Hundley, Heather A.; Biology, School of Science
    Adenosine deaminases that act on RNA (ADARs) are present in all animals and function to both bind double-stranded RNA (dsRNA) and catalyze the deamination of adenosine (A) to inosine (I). As inosine is a biological mimic of guanosine, deamination by ADARs changes the genetic information in the RNA sequence and is commonly referred to as RNA editing. Millions of A-to-I editing events have been reported for metazoan transcriptomes, indicating that RNA editing is a widespread mechanism used to generate molecular and phenotypic diversity. Loss of ADARs results in lethality in mice and behavioral phenotypes in worm and fly model systems. Furthermore, alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases and cancers. In this review, a basic introduction to ADAR structure and target recognition will be provided before summarizing how ADARs affect the fate of cellular RNAs and how researchers are using this knowledge to engineer ADARs for personalized medicine. In addition, we will highlight the important roles of ADARs and RNA editing in innate immunity and cancer biology.