Biochemical Mechanism of Gene Expression Silencing by piRNA-directed PIWI-Clade Argonautes
Authors
Arif, AmenaFaculty Advisor
Phillip D. ZamoreAcademic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
RNA Therapeutics InstituteDocument Type
Doctoral DissertationPublication Date
2021-08-10Keywords
piRNAPIWI
Argonaute
Zinc-Finger
GTSF1
Spermatogenesis
RNAi
miRNAs
siRNAs
Asterix
Evolution
small RNA methylation
non-coding RNAs
small-silencing RNAs
Biochemistry
Biochemistry, Biophysics, and Structural Biology
Molecular Biology
Metadata
Show full item recordAbstract
Argonaute proteins are small DNA/RNA-guided endonucleases found in all domains of life. In animals, small RNAs of length 21–35 nucleotides direct the PIWI-clade of Argonautes to silence complementary target RNAs; these are called PIWI-interacting RNAs (piRNAs). During spermatogenesis in mice, piRNA-guided PIWI proteins, MIWI2, MILI, and MIWI, silence transposons, regulate expression of protein-coding genes and are necessary for fertility. A working endonuclease activity of MIWI and MILI is essential to complete spermatogenesis. Yet, both MIWI and MILI produce weak and slow target cleavage in vitro, thwarting biochemical examination of the silencing step. Here, we find that PIWI proteins require an auxiliary protein to efficiently cleave their targets, unlike any other known Argonaute. Gametocyte Specific Factor 1 (GTSF1) is a conserved zinc-finger protein essential for fertility and piRNA-directed silencing. We show GTSF1 accelerates the pre-steady-state rate of target cleavage by MIWI and MILI; this role of GTSF1 is also preserved in insects. A critical step in GTSF1 mechanism entails binding RNA. GTSF1 allowed detailed kinetic analyses of catalytic PIWIs: they require extensive 3′ complementarity between the guide and target to efficiently cleave them, but this base-pairing also limits turnover. Interestingly, within a species, different PIWI proteins have unique kinetic properties. In sum, our findings provide molecular mechanisms of GTSF1 function and target silencing by PIWIs as well as a useful method for future studies.DOI
10.13028/e70a-dc62Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31386Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/e70a-dc62