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

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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.