Browsing by Subject "RNA-binding protein"

Now showing 1 - 4 of 4
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    Developing Novel Methods to Identify RNA-Associated Mechanisms for Inheritance
    (2020-11) Ettaki, Zacharia Nabil; Aoki, Scott T.; Georgiadis, Millie; Quilliam, Lawrence
    Animals depend on inheriting non-genetic information early in life to grow and develop naturally. This inherited, non-genetic information was previously thought to be limited to DNA modifications and DNA binding proteins. But recent studies have expanded our understanding of inheritance to include RNA and RNA binding proteins. We currently lack methods to identify and enrich for RNA binding proteins that might be involved in providing non-genetic information from mother to daughter cells. Others have developed a method using modified enzyme tags to pulse-label proteins with small molecule fluorescent ligands and follow these proteins as they are inherited by cells. Here I characterized and tested the application of a fluorescent small molecule targeting antibody to enrich for these labeled proteins. I first tested the ability of this antibody to bind to fluorescent ligand-labeled enzymes. I determined that the antibody can efficiently bind to at least one of the labeled enzymes. Second, I determined crystallization conditions for the ligand binding antibody fragment. This thesis sets the stage for structure determination and to test whether this antibody can work in vivo to enrich for RNA binding proteins involved in the delivery of non-genetic information to cells.
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    Modulator-Dependent RBPs Changes Alternative Splicing Outcomes in Kidney Cancer
    (Frontiers Media, 2020) Wang, Yang; Chen, Steven X.; Rao, Xi; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
    Alternative splicing alterations can contribute to human disease. The ability of an RNA-binding protein to regulate alternative splicing outcomes can be modulated by a variety of genetic and epigenetic mechanisms. In this study, we use a computational framework to investigate the roles of certain genes, termed modulators, on changing RBPs’ effect on splicing regulation. A total of 1,040,254 modulator-mediated RBP-splicing interactions were identified, including 137 RBPs, 4,309 splicing events and 2,905 modulator candidates from TCGA-KIRC RNA sequencing data. Modulators function categories were defined according to the correlation changes between RBPs expression and their targets splicing outcomes. QKI, as one of the RBPs influencing the most splicing events, attracted our attention in this study: 2,014 changing triplets were identified, including 1,101 modulators and 187 splicing events. Pathway enrichment analysis showed that QKI splicing targets were enriched in tight junction pathway, endocytosis and MAPK signaling pathways, all of which are highly associated with cancer development and progression. This is the first instance of a comprehensive study on how alternative splicing outcomes changes are associated with different expression level of certain proteins, even though they were regulated by the same RBP. Our work may provide a novel view on understanding alternative splicing mechanisms in kidney cancer.
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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).