Regulation of Argonaute Nuclear Import in Mediating Transgenerational Fertility - PROJECT SUMMARY Nuclear RNA interference (RNAi) is a conserved gene regulatory pathway that is essential for transgenerational fertility and genome stability. Mediated by Argonaute proteins and small RNAs, nuclear RNAi ensures proper regulation of vital processes such as accurate gametogenesis, proper embryonic development, and transposon silencing. Given their critical biological importance, defective and dysregulated Argonaute proteins are associated with various reproductive and developmental disorders, as well as cancer formation and progression. Since the nuclear RNAi pathway and the Argonaute protein structure are highly conserved, we can use C. elegans to investigate these complex human disorders. Our long-term goal is to use C. elegans to characterize the nuclear RNAi pathway, which will provide insight into the molecular biology that drives these human disorders and how it affects fertility, development, and genome integrity. There remain critical outstanding questions regarding the nuclear RNAi pathway. Firstly, Argonaute nuclear import remains poorly characterized, even though it is imperative for nuclear RNAi. Secondly, the biological function of the N-terminal intrinsically disordered regions (IDRs) of Argonaute proteins remains elusive. Thirdly, post-translational modifications (PTMs) and their role in regulating nuclear Argonaute function are not well characterized. The objective of this proposal is to determine the regulation and molecular mechanism of Argonaute nuclear import that mediates transgenerational fertility. The proposal will address the central hypothesis that the N-terminal IDR of nuclear Argonaute protein HRDE-1, is essential for small RNA-loading and that PTMs regulate HRDE-1 function and nuclear import. Aim 1 will elucidate the biological function of the HRDE-1 N-terminal IDR, using innovative experiments such as CRISPR editing, small RNA-binding assays and sequencing, and fluorescence imaging. Importantly, we will directly determine the effect on transgenerational fertility and RNAi inheritance in these mutants with phenotypic assays. Aim 2 will characterize the role of PTMs in regulating HRDE-1 sRNA binding and nuclear import, utilizing the experiments listed in Aim 1 as well as the phenotypic assays. Aim 3 will determine the factors and pathways that mediate HRDE-1 nuclear import. Potential nuclear import factors have been identified and preliminary results have verified two candidates to be true HRDE-1 nuclear import factors. We will continue screening other candidates with RNAi gene knockdown and fluorescence imaging experiments. The physical interaction of HRDE-1 and the nuclear import factors will be determined with co-immunoprecipitations. The expected results of this proposal will determine the biological function of HRDE-1 N-terminal IDR, as well as identify the molecular mechanism and factors that mediate HRDE-1 nuclear import. The overall impact of this research is that it will provide valuable molecular insight into the conserved nuclear RNAi pathway, which will shed light on how dysregulation of the pathway can lead to devastating human disorders.
