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Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2 . We show that this unannotated AFE isoform of Aim2 is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5'UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2 ., Competing Interests: ER, PJ, SC, MC, VS, RS, BS, RA, MJ, SC, SC No competing interests declared, MA holds options in Oxford Nanopore Technologies (ONT), is a paid consultant to ONT, received reimbursement for travel, accommodation and conference fees to speak at events organized by ONT,received research funding from ONT and is an inventor on 11 UC patents licensed to ONT (6,267,872, 6,465,193, 6,746,594, 6,936,433, 7,060,50, 8,500,982, 8,679,747, 9,481,908, 9,797,013, 10,059,988, and 10,081,835), AB received reimbursement for travel, accommodation and conference fees to speak at events organized by Oxford Nanopore Technologies (ONT), (© 2021, Robinson et al.)

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

The innate immune system protects against infections by initiating an inducible inflammatory response. NF-κB is one of the critical transcription factors controlling this complex response, but some aspects of its regulation remain unclear. For example, although long non-coding RNAs (lncRNAs) have been shown to critically regulate gene expression, only a fraction of these have been functionally characterized, and the extent to which lncRNAs control NF-κB expression is unknown. Here, we describe the generation of a GFP-based NF-κB reporter system in immortalized murine bone marrow-derived macrophages (iBMDM). Activation of this reporter, using Toll-like receptor ligands, resulted in GFP expression, which could be monitored by flow cytometry. We also established a CRISPR/Cas9 gene deletion system in this NF-κB reporter line, enabling us to screen for genes that regulate NF-κB signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-κB signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting IκBα degradation in the cytoplasm. For lincRNA-AK170409, we provide evidence that this nuclearly-localized lincRNA regulates a number of inflammation-related genes. In conclusion, we have established an NF-κB-GFP iBMDM reporter cell line and a line that stably expresses Cas9. Our approach enabled the identification of lincRNA-Cox2 and lincRNA-AK170409 as NF-κB regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)