Myelodysplastic syndrome-associated spliceosome gene mutations enhance innate immune signaling Genes encoding spliceosome components including SF3B1, U2AF1, and SRSF2, are frequently somatically mutated in myelodysplastic syndromes (MDS), other hematologic malignancies, and solid tumors. 1 Typically these are mutually exclusive, heterozygous, missense, hotspot mutations that result in neomorphic or gain-offunction splicing phenotypes. These mutations alter splicing of many genes; however, overlap among the different splicing factors is limited. Thus, a common mechanism by which spliceosome mutations contribute to disease is suspected but has remained elusive. We previously demonstrated that inhibiting any of five different spliceosome genes (SF3B1, SF3A1, SF3A2, SF3A3, EFTUD2) in mouse or human macrophages reduces inflammatory cytokine production induced by multiple Toll-like receptor (TLR) agonists including the TLR4 agonist lipopolysaccharide (LPS). 2-5 Although these genes encode essential spliceosome components, partial gene knockdown (approx. 80%) reduces LPS-induced inflammatory cytokine production without affecting viability or phagocytosis. 2-5 Hence, innate immunity may be particularly sensitive to spliceosome perturbation. These observations suggest that MDS-associated spliceosome mutations might enhance innate immunity,

SUMMARY Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion, and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b, and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion.

Alveolar macrophages serve as central orchestrators of inflammatory responses in the lungs, both initiating their onset and promoting their resolution. However, the mechanisms that program macrophages for these dynamic responses are not fully understood. Over 95% of all mammalian genes undergo alternative pre-mRNA splicing. While alternative splicing has been shown to regulate inflammatory responses in macrophages in vitro, it has not been investigated on a genome-wide scale in vivo. Here we used RNAseq to investigate alternative pre-mRNA splicing in alveolar macrophages isolated from lipopolysaccharide (LPS)-treated mice during the peak of inflammation and during its resolution. We found that lung inflammation induced substantial alternative pre-mRNA splicing in alveolar macrophages. The number of changes in isoform usage was greatest at the peak of inflammation and involved multiple classes of alternative pre-mRNA splicing events. Comparative pathway analysis of inflammation-induced changes in alternative pre-mRNA splicing and differential gene expression revealed overlap of pathways enriched for immune responses such as chemokine signaling and cellular metabolism. Moreover, alternative pre-mRNA splicing of genes in metabolic pathways differed in tissue resident vs. recruited (blood monocyte-derived) alveolar macrophages and corresponded to changes in core metabolism, including a switch to Warburg-like metabolism in recruited macrophages with increased glycolysis and decreased flux through the tricarboxylic acid cycle.

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of and, two genes that function in multiple TLR pathways. The and genes produce long proinflammatory mRNAs (MyD88 and IRAK1) and shorter anti-inflammatory mRNAs (MyD88 and IRAK1c). We quantified mRNA encoding inflammatory cytokines and and isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in and alternative splicing observed in previous in vitro studies, there were differences in how and alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches.