ORCID ID

0000-0002-4902-5167

Publication Date

2021-08-10

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

RNA Therapeutics Institute

First Thesis Advisor

Phillip D. Zamore

Keywords

piRNA, PIWI, Argonaute, Zinc-Finger, GTSF1, Spermatogenesis, RNAi, miRNAs, siRNAs, Asterix, Evolution, small RNA methylation, non-coding RNAs, small-silencing RNAs

Abstract

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-dc62

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Saturday, September 09, 2023

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