Biochemistry in situ to determine inheritance of RNA-protein complexes

Biochemistry in situ to determine inheritance of RNA-protein complexes
The long-term goal is to elucidate the molecular mechanisms how macromolecular RNA-protein complexes
transmit information to future generations of cells and progeny. We classically think of DNA and DNA
modifications as the only information inherited between cells. Recent work demonstrates that RNA and RNA-
binding proteins are also inherited and that these proteins have functions in organism development and
immunity. My lab aims to identify which RNA binding proteins are inherited, determine the macromolecular
organization of inherited RNA-protein complexes, and discover the molecular purpose of inheriting these protein
complexes. This interest in inherited RNA binding proteins currently extends to investigating RNA-protein
complexes that form multi-component RNA-protein granules, or biomolecular condensates. Much of my previous
work centered on characterizing the structural organization of P granules, an inherited RNA-protein granule
necessary for C. elegans nematode germ cell development. The next five years will focus on understanding the
molecular mechanisms how RNA-protein complexes are inherited across cell generations and from parent to
progeny. Which RNA binding proteins are inherited, and which cells inherit these proteins? What protein
attributes are required for inheritance? Our ability to investigate these questions is currently limited by available
methods to track protein components in multicellular organisms. My lab seeks to label and follow maternal
proteins in the authentic germline tissue of C. elegans, a proven model organism to study basic questions in
animal development. Established single gene editing methods, robust imaging capabilities, and short
generational time make C. elegans an ideal multicellular animal to identify the functions of maternal proteins
inherited across generations. Modified enzyme tags now allow us to pulse label proteins with covalently bound
ligands and chase these labeled proteins over time. This in vivo pulse-chase method has been used to follow
chromatin remodeling in cell culture and protein stability in mouse tumors. Our preliminary results demonstrate
that we are able to use in vivo pulse chase to track histone protein stability in worm germline tissue under different
nutrient conditions. The current goal is to use in vivo pulse chase in C. elegans to visualize the stability of
maternal germline RNA binding proteins through germ cell development and track these proteins as they are
inherited from mother to progeny. First, we will pulse-label a P granule assembly protein and test how granule
formation and protein quantity affect its inheritance. Second, we will pulse-chase maternal germline Argonautes,
a family of RNA regulatory enzymes, to identify which Argonautes are inherited by progeny, what tissues inherit
them, and what protein attributes are necessary for tissue-specific inheritance. Collectively, this work will redefine
our concept of maternal inheritance and elucidate criteria for the inheritance of specific RNA binding proteins. In
vivo pulse-chase in C. elegans will provide a foundation to discover novel maternally inherited proteins
associated with gene regulation for development, immunity, and beyond.
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Grant Number: 5R35GM142691-05
NIH Institute/Center: NIH
Principal Investigator: Scott Aoki