Macrophage development and activation involve coordinated intron retention in key inflammatory regulators

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response

The role of RNA modifications in macrophages

RNA modifications and effector RNA binding proteins regulate gene expression in most biological processes. Amongst RNA modifications are N6-methyladenosine (m6A), which results from methylation of adenosine residues by the METTL3-METTL14 methyltransferase complex, and RNA 5-hydroxymethylcytosine (5hmC), which arises from oxidation of 5-methylcytosine by the Ten-Eleven Translocation (TET) enzymes. While m6A is established as a versatile regulator of RNA metabolism in various biological contexts, the functions of RNA 5hmC are unknown. Despite some evidence linking RNA modifications to immunity, their implications in macrophage development and functions are unclear. Since macrophages are central effectors of the innate immune response and are heavily involved in chronic inflammation, understanding the molecular pathways behind macrophage-mediated responses is critical to developing new treatments for inflammatory disorders. In this thesis, I present novel insights into m6A- and 5hmC-mediated gene expression regulation in macrophages. I found that m6A and 5hmC occur together in transcripts with key roles in macrophage biology and that METTL3 and TET enzymes are involved in macrophage differentiation. I also identified specific transcripts regulated by these enzymes in a cellular-context-dependent manner. Furthermore, I discovered that METTL3 regulates inflammasome activation, a critical event that triggers the innate immune response. I demonstrate that METTL3 depletion protects mice from septic shock-induced death and attenuates NLRP3 hyperactivation in primary cells isolated from patients suffering from an NLRP3 inflammasome-driven autoinflammatory disease. Mechanistically, I found that METTL3-mediated m6A methylation promotes NLRP3 translation. This thesis extends current knowledge on RNA modifications in macrophages and the immune response and establishes RNA modification regulatory proteins as potential targets for therapeutic intervention in inflammatory diseases

A multiomics dataset for the study of RNA modifications in human macrophage differentiation and polarisation

Abstract RNA modifications have emerged as central regulators of gene expression programs. Amongst RNA modifications are N6-methyladenosine (m6A) and RNA 5-hydroxymethylcytosine (5hmC). While m6A is established as a versatile regulator of RNA metabolism, the functions of RNA 5hmC are unclear. Despite some evidence linking RNA modifications to immunity, their implications in gene expression control in macrophage development and functions remain unclear. Here we present a multi-omics dataset capturing different layers of the gene expression programs driving macrophage differentiation and polarisation. We obtained mRNA-Seq, m6A-IP-Seq, 5hmC-IP-Seq, Polyribo-Seq and LC-MS/MS data from monocytes and resting-, pro- and anti-inflammatory-like macrophages. We present technical validation showing high quality and correlation between samples for all datasets, and evidence of biological consistency of modelled macrophages at the transcriptomic, epitranscriptomic, translational and proteomic levels. This multi-omics dataset provides a resource for the study of RNA m6A and 5hmC in the context of macrophage biology and spans the gene expression process from transcripts to proteins

Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC-3 prostate cancer cell lines, we showed that chemical or shRNA-mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2-mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC-3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down-regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2-mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer

Intron retention enhances gene regulatory complexity in vertebrates

Background: While intron retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic