Determination of the importance of MNDA and the potential implication of other PYHIN factors in (pre-)mRNA processing

背景：髓系核分化抗原（MNDA）是PYHIN家族的一种应激诱导因子。人PYHIN因子MNDA、AIM 2、IFI 16和IFIX位于细胞核和细胞质中，并且已知影响细胞凋亡。MNDA是促进髓样和淋巴样细胞中凋亡进展的核-微电子回路的中心介质。当它在细胞质中积累时，MNDA促进抗凋亡因子MCL-1的降解。我们最近发现，核MNDA也可以影响细胞凋亡，因为它与RNA结合复合物相互作用，并在应激诱导MNDA负调节Mcl-1和Bcl-2转录产物。PYHIN因子均具有高度同源的Pyrin和HIN 200结构域。人PYHIN因子可通过其HIN 200结构域结合双链DNA。是否所有的人PYHIN因子都可以通过控制RNA的产生/加工来影响细胞功能尚不清楚。 计划长期目标：定义控制转录机器如何响应各种生理和压力信号的机制。 未来5年的具体目标：确定MNDA和其他PYHIN因子是否可以影响和促进应激诱导后转录组的快速修饰。 假设：MNDA和其他PYHIN因子通过控制特异性（前）mRNA的合成和加工参与应激反应。研究方法：（1）为了确定在正常与应激条件（顺铂暴露）下MNDA靶向的转录产物，将进行RNA免疫沉淀测序（RIP-seq）测定。本研究将使用淋巴系造血细胞。我们还将通过RNA-Seq和RT-qPCR确定MNDA是否影响这些转录物的产生。(2)我们发现MNDA蛋白与参与转录产物的产生、加工和转运的蛋白质相互作用。我们将定义MNDA蛋白相互作用组在遗传毒性应激前后的变化。将进行互补串联亲和纯化（TAP-标签）和BioID试验，以鉴定（LC-MS/MS分析）正常与强制降解条件下MNDA蛋白相互作用组的精确变化。这些试验将得到免疫共沉淀、免疫荧光和分子分级FPLC试验的支持。还将进行疑似影响MNDA功能的MNDA相互作用配偶体的敲低/敲除。(3)PYHIN因子的Pyrin和HIN 200结构域非常相似，因此，我们将确定其他PYHIN因子是否类似地影响mRNA。 PYHIN因子的核重要性和功能仍然不清楚。在这个5年的任期中描述的研究是我们的NSE研究计划的基础，因为它将显着推进我们对这些应激反应因子的生物学知识，并将提供有关适应细胞应激所需的转录组可塑性控制机制的信息。

Background: The Myeloid Nuclear Differentiation Antigen (MNDA) is a stress inducible factor of the PYHIN family. The human PYHIN factors, MNDA, AIM2, IFI16 and IFIX, are located in the nucleus and cytoplasm, and are known to influence apoptosis. MNDA is a central mediator of a nucleus-mitochondrion circuit that promotes progression of apoptosis in myeloid and lymphoid cells. When it accumulates in the cytoplasm, MNDA promotes degradation of the anti-apoptotic factor MCL-1. We recently found that the nuclear MNDA can also influence apoptosis since it interacts with RNA binding complexes and upon stress induction MNDA negatively regulates the Mcl-1 and Bcl-2 transcription products. The PYHIN factors are all characterized by highly homologous Pyrin and HIN200 domains. The human PYHIN factors can bind double stranded DNA by their HIN200 domain. Whether all human PYHIN factors can influence cellular functions by the control of RNA production/processing is not known. Program long-term goals: To define the mechanisms that govern how the transcriptional machinery responds to various physiological and stress signals. Specific aim for the next 5 years: To determine whether MNDA and other PYHIN factors can influence and favor rapid modifications of the transcriptome upon stress induction. Hypothesis: MNDA and other PYHIN factors participate to stress response by controlling synthesis and processing of specific (pre-)mRNA. Methods: (1) To define the transcription products targeted by MNDA in normal vs stress conditions (cisplatin exposure), the RNA immunoprecipitation-sequencing (RIP-seq) assay will be performed. Hematopoietic cells of the lymphoid lineage will be utilised for this study. We will also determine whether MNDA influences the production of these transcripts by RNA-Seq and RT-qPCR. (2) We found that MNDA protein interacts with proteins involved in production, processing and transport of transcription products. We will define the variations of the MNDA protein interactome before and after genotoxic stress. The complementary Tandem Affinity Purifications (TAP-tag) and BioID assays will be performed to identify (LC-MS/MS analyses) precise variations in the MNDA protein interactome in normal vs stress condition. These assays will be supported by co-immunoprecipitation, immunofluorescence and size fractionation FPLC assays. The knockdown/knockout of MNDA interacting partners suspected to affect MNDA function will also be made. (3) Pyrin and HIN200 domains of the PYHIN factors are very similar and thus, we will define if other PYHIN factors similarly influence mRNA. The nuclear importance and function(s) of PYHIN factors is still poorly defined. The study described in this 5-year term is fundamental to our NSE research program, as it will significantly advance our knowledge on the biology of these stress response factors and will provide information on mechanisms controlling transcriptome plasticity required for adaptation to cellular stress.