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    Unraveling the Roles of C. elegans Vasa Homologs, GLH Proteins, in Argonaute Pathway Specificity and Transcriptome Surveillance

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    Dissertation Siyuan Dai 230525.pdf
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    Dissertation
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    Authors
    Dai, Siyuan
    Faculty Advisor
    Craig Mello
    Academic Program
    Interdisciplinary
    UMass Chan Affiliations
    RNA Therapeutics Institute
    Document Type
    Doctoral Dissertation
    Publication Date
    2023-05-25
    Keywords
    DEAD-box ATPase
    Vasa Homologs
    Argonaute pathway
    small-RNA biogenesis
    transgenerational epigenetic inheritance
    Germline nuage
    
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    Abstract
    Epigenetic regulation of gene expression empowers organisms to alter phenotypic information without affecting DNA sequence. Germline Argonaute proteins, complexed with their cognate small-RNAs, are essential for transcriptome surveillance and maintenance of heritable gene silencing. In C. elegans, PIWI Argonaute PRG-1 employs piRNAs to screen thousands of germline transcripts through microRNA-like base pairing. Upon target recognition, RNA-directed RNA polymerases (RdRPs) are recruited to generate abundant antisense small RNAs (22G-RNAs), which are subsequently loaded onto worm-specific Argonautes (WAGOs) to establish long-term epigenetic memory. Both small RNA amplification and Argonaute surveillance are thought to occur within perinuclear liquid-like condensates called nuage or P granules. The precise mechanism by which the nearly one million different perinuclear Argonaute/guide complexes engage their targets to mediate gene regulation remain unclear. In this dissertation, we explore the functions of a family of VASA homologs, Germ Line Helicases (GLHs) in diverse germline small-RNA pathways. Our genetic and biochemical investigations reveal that these perinuclear-localized DEAD-box proteins engage germline transcripts and promote piRNA- and RNA interference-mediated transgenerational silencing in C. elegans. We provide evidence that GLH proteins scaffold multiple Argonautes responsible for epigenetic silencing, competing with paralogs for direct binding to target mRNAs. Additionally, GLHs enhance the specificity of Argonuate pathways by preventing WAGO/22G-RNA misrouting. Through examining mutants without severe P-granule disruptions, we sought to separate GLH-1 scaffolding and enzymatic functions. We found that the GLH-1 ATPase cycle promotes RNA duplex unwinding and the biogenesis of WAGO-bound 22G-RNAs while many P-granule components remain properly localized. Moreover, we show that GLH-1 N-terminal domains containing both FG repeats and zinc fingers are crucial for binding Argonautes and RNA substrates. Taken together, our findings support a model in which Argonautes recruit GLH proteins for target mRNA binding and retention, enabling GLHs to regulate pathway-specific small-RNA signals and transgenerational inheritance.
    DOI
    10.13028/33vp-q404
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/52128
    Rights
    Copyright © 2023 Siyuan Dai
    Distribution License
    All Rights Reserved
    ae974a485f413a2113503eed53cd6c53
    10.13028/33vp-q404
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