RNA Structure Probing Approaches to Investigating Pro-Inflammatory RNA Helicases - PROJECT SUMMARY The importance of the extended family of 58 human DEAD-/DEAH-box RNA helicases to human health and disease has escalated, owing to their emerging roles in innate immunity and inflammation, neurological disease, hematological disorders, and cancer. These structurally related enzymes cleave ATP to trigger binding and unbinding to RNA targets. Helicases locally destabilize RNA structure through transient interactions, unwinding and/or remodeling base-paired regions. Helicases have short-lived, often sequence-independent interactions with their targets. Because these interactions are difficult to measure, especially in cells, the functions of many helicases are therefore poorly defined. Here, we propose to address this gap in knowledge by identifying the RNA substrates of helicases and the functional regions they remodel in vivo. Specifically, we recently developed Co-transcriptional Structure Tracking (CoSTseq), which uses chemical probing with DMS to detect nascent RNA structure formation during transcription elongation. CoSTseq can be performed in parallel with DMS-MaPseq, which reveals structure in steady-state, mature RNA. We previously measured reactivity differences between nascent and mature rRNA and discovered effects of regulatory helicases on nascent rRNA, by comparing datasets from yeast strains harboring helicase mutations or deletions. Thus, chemical probing of nascent and mature RNA combined with helicase depletion/mutagenesis represents a powerful and general strategy for understanding RNA helicases. We expect our approach will determine (i) the RNA substrates they remodel and (ii) the functional RNA regions in which structure is altered (e.g. 5’or 3’UTRs, exons, introns). Recently, RNA helicases have emerged as pro-inflammatory regulators with roles in pre-mRNA splicing, RNA export and stability, and mRNA translation, yet how their helicase activities contribute to inflammation is mostly unknown. Mammalian DDX3X is associated with numerous diseases, is cytoplasmic and nuclear, and promotes translation. The yeast homolog Ded1 also promotes splicing (our preliminary data). DDX3X binds to the NLRP3 inflammasome and increases its activation, while DDX3X sequestration in stress granules (SGs) reduces activation. Our working model is that DDX3X remodels nascent and mRNA 5’UTRs as well as mRNAs present in SGs, signifying both cellular and molecular mechanisms of regulation. However, a clear list of nascent or mRNA targets and the regions DDX3X remodels is lacking. Our three specific aims combine to determine direct roles of DDX3X in RNA processing and RNA folding during macrophage activation in vivo. Aim 1 will investigate the activities of Ded1 in yeast to refine expectations, workflows and data analysis. Aim 2 will investigate resting and activated human macrophages in culture, using rapid depletion of DDX3X to identify nascent and mRNA targets related to the inflammatory response. Aim 3 will ask if changes in RNA folding can be detected by chemical inhibitors of arginine dimethylation, which should alter the interactions of DDX3X with a key SG protein, TDRD3. These inhibitors could have the properties of an anti-inflammatory therapeutic agent.
