Project description:We investigated the role of RNMT in T cells using an Rnmt conditional knockout mouse model. We report that the mRNA cap methyltransferase, RNMT, supports naïve T cell survival and activation-induced proliferation. We demonstrate that RNMT has gene-specific impacts in T cells, selectively regulating expression of terminal polypyrimidine tract (TOP) mRNAs which are targets of the m7G-cap binding protein LARP1. These LARP1 eCLIP experiments determine the RNA binding sites of LARP1 in naive CD4 T cells from Rnmt cKO and control mice.
Project description:CD4+ T cells are critical components in the human immune system. They produce cytokines to fight against pathogens and abnormal cells and stimulate other cells, such as B cells, macrophages, and neutrophils, to generate an immune response.
Naive CD4+ T cells are precursor cells that can differentiate into T helper - 1, - 2, - 17 (Th1, Th2, Th17) and regulatory T cells (Tregs) subtypes based on the type of pathogens or disease. The naive CD4+ T cell model consists of 5179 reactions, 3153 metabolites, and 1055 genes. Together with Th1, Th2, and Th17 models, the naive CD4+ T cell model helped identify drug targets and repurposable drugs against autoimmune diseases.
Project description:RNA sequencing of the chromatin associated RNA and nucleoplasm associated RNA of Naive CD4+ T cells to identify novel chromatin associated RNAs containing TEs. RNA sequencing of Naive CD4+ T cells or Activated Naive CD4+ T cells treated with Scr or LINE1 antisense oligonucleotides (ASO).
Project description:Naive CD4+ T cells are the common precursors of multiple effector and memory T cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy.<br>Periodate oxidation and aniline-catalyzed oxime ligation (PAL) technology was applied with subsequent quantitative LC-MS/MS (PAL-qLC-MS/MS) to generate a dataset describing the surface proteome of human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins, of which 24 were previously not known to be expressed on human naive CD4+ T cells or have no defined role within T cell activation. To independently confirm the proteomic dataset and to analyse the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous dataset, resulting in 229 surface proteins which are expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation and predicted subcellular localization, and correlated the proteomics result with this transcriptional dataset.<br>This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments.
Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis
Project description:This project focuses on examining the differences in the global CD4+ T cell proteome between cells lacking the negative regulators of eIF4E( eIf4E-Binding Proteins 1/2) and WT naive CD4+ T cells both prior and following TCR stimulation.