The NIP proteins: Inhibitors of nuclear RNA interference

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Background.***The RNA interference (RNAi) pathways instigate gene silencing through transcriptional and post-transcriptional mechanisms. SiRNAs of endogenous and exogenous origins are loaded onto specialized cytoplasmic and nuclear Argonautes, which in turn direct gene-silencing complexes. Loading of nuclear Argonautes results in their nuclear translocation, and in recruitment of effector complexes, which mediate the deposition of silencing chromatin marks on target loci. We discovered the family of uncharacterized Nuclear Argonaute-Interacting Proteins (NIP-1, NIP-2 will be examined), which stably interact with NRDE-3. NIP proteins encode a conserved P-loop containing nucleoside triphosphate hydrolase (NTPase) domain. Loss-of-function alleles specifically enhance nuclear RNAi and NIP-1 overexpression results in refraction, while having little effect on cytoplasmic RNAi targets. NIP-1 interacts with unloaded NRDE-3, and does not interact with small RNAs. ******Hypothesis.*** Argonautes are limiting for the silencing activities in the RNAi pathways. As such, we hypothesize that NIP-1/2 proteins repress nuclear RNAi by preventing the loading of nuclear Argonautes and their consequent translocation to the nucleus, thus allowing direct control on the output of nuclear RNAi.******Specific Aims:***1- To determine the effect of NIP-1/2 on endogenous nuclear RNAi targets, we will immunoprecipitate the nuclear Argonaute NRDE-3 and sequence its associated siRNAs in WT, nip-1/2 mutants, and in over-expression strains. SiRNAs will be quantified using next-generation sequencing, and changes in expression of endogenous target loci will be validated. We will further test the impact of nip-1/2 on the odr-1 adaptation cascade, a physiological cascade directly regulated by nuclear RNAi***2- To understand how NIP-1/2 proteins inhibit nuclear RNAi, we will map their physical interactions with NRDE-3, and test their significance by engineering disrupting alleles in vivo. We will further probe the interplay between the NLS of NRDE-3 and NIP-1/2 interactions. Finally, we will perform a comparative approach of NIP-1 and NIP-2 interactions using AP-MS/MS.***3- To examine how NIP-1/2 interaction with NRDE-3 is regulated, we will image NRDE-3 sub-cellular localization in nip-1/2 mutants, and in over-expression strains. We furthermore will test the significance of the NTPase domain of NIP-1/2 for NRDE-3 localization, function and interactions.******Significance.***RNAi pathways functionally intersect with chromatin in the nucleus in organisms ranging from S. pombe to human. Elucidating the principles underlying nuclear RNAi mechanisms will enable entryways into physiological and pathological epigenomes.