The NIP proteins: Inhibitors of nuclear RNA interference

背景 RNA干扰（RNAi）途径通过转录和转录后机制引发基因沉默。内源性和外源性来源的siRNA被加载到专门的细胞质和核Argonaute上，这反过来又指导基因沉默复合物。核Argonaute的加载导致其核易位，并募集效应复合物，其介导沉默染色质标记在靶基因座上的沉积。我们发现了未表征的核Argonaute-Interacting蛋白家族（NIP-1，NIP-2将被检查），其与NRDE-3稳定地相互作用。NIP蛋白编码一个保守的含有三磷酸核苷水解酶（NTH）结构域的P环。功能丧失等位基因特异性地增强核RNAi和NIP-1过表达导致屈光，而对细胞质RNAi靶标几乎没有影响。NIP-1与未加载的NRDE-3相互作用，并且不与小RNA相互作用。 假说. Argonaute限制了RNAi途径中的沉默活性。因此，我们假设NIP-1/2蛋白通过阻止核Argonaute的装载及其随后的易位到核来抑制核RNAi，从而允许直接控制核RNAi的输出。 具体目标： 1-为了确定NIP-1/2对内源性核RNAi靶标的作用，我们将免疫沉淀核Argonaute NRDE-3并在WT、nip-1/2突变体和过表达菌株中对其相关siRNA进行测序。将使用下一代测序对siRNA进行定量，并验证内源性靶基因座表达的变化。我们将进一步测试nip-1/2对odr-1适应级联的影响，odr-1适应级联是一种由核RNAi直接调节的生理级联。 2-为了了解NIP-1/2蛋白如何抑制核RNAi，我们将绘制它们与NRDE-3的物理相互作用，并通过体内工程破坏等位基因来测试它们的意义。 我们将进一步探讨NRDE-3和NIP-1/2相互作用的NLS之间的相互作用。最后，我们将使用AP-MS/MS对NIP-1和NIP-2相互作用进行比较。 3-为了研究NIP-1/2与NRDE-3的相互作用是如何被调节的，我们将对nip-1/2突变体和过表达菌株中的NRDE-3亚细胞定位进行成像。我们还将测试NIP-1/2的NTR结构域对NRDE-3定位、功能和相互作用的意义。 意义 RNAi途径在功能上与细胞核中的染色质交叉，从S.从粟酒到人类阐明核RNAi机制的基本原理将使进入生理和病理表观基因组成为可能。

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.