The NIP proteins: Inhibitors of nuclear RNA interference

背景资料。 RNA干扰(RNAi)途径通过转录和转录后机制诱导基因沉默。内源和外源的siRNAs被装载到专门的细胞质和核精氨酸上，这反过来又指导了基因沉默复合体。核精氨酸的负载导致了它们的核移位和效应复合体的募集，这些效应复合体介导了靶基因上沉默染色质标记的沉积。我们发现了一个未鉴定的核精氨酸相互作用蛋白家族(NIP-1，NIP-2将被检测)，它们与NRDE-3稳定地相互作用。NIP蛋白编码一个保守的含有核苷三磷酸水解酶(NTPase)结构域的P-环。功能缺失等位基因特异性地增强核RNAi和NIP-1的过度表达导致屈光，而对细胞质RNAi靶点几乎没有影响。NIP-1与卸载的NRDE-3相互作用，而不与小RNA相互作用。 假设。 宇航员正在限制RNAi途径中的沉默活动。因此，我们假设NIP-1/2蛋白通过防止核精氨酸的负载和随之而来的核移位来抑制核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靶标的影响，我们将免疫沉淀核Argavite NRDE-3，并对其相关的siRNAs在WT、NIP-1/2突变体和高表达菌株中进行测序。SiRNA将使用下一代测序进行量化，并将验证内源性靶基因表达的变化。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的物理相互作用图，并通过在体内工程干扰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的相互作用进行比较。 3-为了研究NIP-1/2与NRDE-3的相互作用是如何调节的，我们将在NIP-1/2突变体和高表达菌株中成像NRDE-3的亚细胞定位。我们将进一步测试NIP-1/2的NTPase结构域对NRDE-3的定位、功能和相互作用的意义。 意义重大。 在从S.pombe到人类的各种生物中，RNAi途径在功能上与细胞核中的染色质相交。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的途径成为可能。

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.