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家族的应激诱导因子。人类PYHIN因子MNDA、AIM2、IFI16和iFIX位于细胞核和细胞质中，并被认为影响细胞凋亡。MNDA是核-线粒体回路的中心介质，促进髓系细胞和淋巴样细胞的凋亡进程。当MNDA积聚在细胞质中时，它会促进抗凋亡因子MCL-1的降解。我们最近发现，核MNDA还可以影响细胞凋亡，因为它与RNA结合复合体相互作用，在应激诱导下，MNDA负调控Mcl-1和Bcl2转录产物。PYHIN因子都具有高度同源的PYHIN和HIN200结构域。人PYHIN因子可通过其HIN200结构域与双链DNA结合。目前尚不清楚是否所有的人类PYHIN因子都可以通过控制RNA的生产/加工来影响细胞功能。 计划的长期目标：定义控制转录机制如何对各种生理和压力信号做出反应的机制。 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有利于转录组在应激诱导时的快速修改。 假设：MNDA和其他PYHIN因子通过控制特定(前)mRNA的合成和处理参与应激反应。 方法：(1)采用RNA免疫沉淀-测序(RIP-seq)方法，确定MNDA在正常和应激条件下(顺铂暴露)靶向转录产物。这项研究将利用淋巴系的造血细胞。我们还将通过RNA-Seq和RT-qPCR来确定MNDA是否影响这些转录本的生产。(2)我们发现MNDA蛋白与参与转录产物的生产、加工和运输的蛋白相互作用。我们将确定MNDA蛋白相互作用组在基因毒性应激前后的变化。互补串联亲和纯化(TAP-Tag)和BioID分析将被用来识别(LC-MS/MS分析)MNDA蛋白相互作用组在正常和应激条件下的精确变异。这些检测将得到免疫共沉淀、免疫荧光和粒度分级FPLC检测的支持。还将对怀疑影响MNDA功能的MNDA互动伙伴进行击倒/敲除。(3)PYHIN因子的PYRIN和HIN200结构域非常相似，因此，我们将确定是否有其他PYHIN因子类似地影响mRNA。 PYHIN因子的核重要性和功能(S)仍然不清楚。这项为期5年的研究是我们NSE研究计划的基础，因为它将极大地促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.