(VP085) POST-TRANSCRIPTIONAL CONTROL OF RIPK1 IN MACROPHAGE INFLAMMATION AND NECROPTOSIS

Background: Receptor-interacting protein kinase 1 (RIPK1) is a major upstream mediator of inflammation and cell death. These pathways are key to inflammatory diseases like atherosclerosis, where macrophages play an important role in their progression. Closely linked to the expression of downstream genes, long non-coding RNAs (lncRNAs) are critical to controlling cellular processes in health and disease. As post-transcriptional regulatory mechanisms for RIPK1 are largely unknown, this project seeks to study Ripk1 mRNA interactions with relevant lncRNAs, along with the stability of Ripk1 mRNA and RIPK1 protein under various conditions. Distinct lncRNAs are hypothesized to interact with Ripk1 mRNA in mouse macrophages undergoing inflammation and necroptosis. Both Ripk1 mRNA and RIPK1 protein stability play important roles in post-transcriptionally regulating RIPK1 activity in normal and inflammatory environments.

METHODS AND RESULTS: We implemented a novel RNA pull-down procedure using biotinylated antisense oligonucleotides and streptavidin magnetic beads to capture Ripk1 mRNA and attached lncRNAs for next-generation sequencing. Through differential expression analysis, we discovered significant upregulation of known lncRNA AC125611 and novel lncRNA MSTRG.5894.1 in Ripk1-targeted samples subject to inflammation. MSTRG.7477.1 was also upregulated during necroptosis, while MSTRG.5684.5 was upregulated during inflammation and necroptosis. GapmeR-mediated knockdowns of AC125611 and MSTRG.5684.5 under inflammatory conditions resulted in decreased Ripk1 mRNA expression and RIPK1 protein expression, respectively. Meanwhile, MSTRG.7477.1 knockdowns were notably connected to less RIPK1 at both the mRNA and protein levels. Using transcription and translation inhibitors, we also determined that both Ripk1 mRNA and RIPK1 protein are relatively unstable with half-lives of approximately 3 h. Their turnover in macrophages is further influenced by the timing and duration of inflammation.

Conclusion: Our research advances the current understanding of RIPK1 regulation by focusing on Ripk1 mRNA-lncRNA associations and turnover of its mRNA and protein in macrophages, paving the way for future investigations into their capacity to act as therapeutic targets.