Identification of LARP1 targets in naive CD4 T cells [eCLIP]. Identification of LARP1 targets in naive CD4 T cells [eCLIP]

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. Overall design: LARP1 seCLIP (single end cross linking immunoprecipitation- an enhanced iCLIP method) libraries were prepared from naïve CD4 T cells magnet sorted from mouse lymph nodes. Proteins were crosslinked to RNA using UV light prior to cell lysis, RNA was partially digested, then LARP1-crosslinked RNA immunoprecipitated and used to prepare libraries. Here we first compared three different antibodies for LARP1 seCLIP in CD4 T cells, then selected one antibody to prepare libraries from Rnmt fl/fl (control) and Rnmt fl/fl CD4cre+ (Rnmt cKO) CD4 T cells. Each experiment has libraries prepared with a LARP1 antibody, an isotype control antibody, and a size matched input control.

本研究采用Rnmt条件性敲除（conditional knockout, cKO）小鼠模型，探究了RNMT在T细胞中的生物学功能。本研究证实，mRNA帽甲基转移酶（mRNA cap methyltransferase）RNMT可维持初始T细胞（naïve T cell）的存活与激活诱导增殖（activation-induced proliferation）。本研究进一步证实，RNMT在T细胞中具有基因特异性调控作用，可选择性调控末端多嘧啶tract（terminal polypyrimidine tract, TOP）mRNA的表达，此类mRNA为m7G帽结合蛋白LARP1（m7G-cap binding protein LARP1）的靶标。

本项LARP1单端交联免疫沉淀（single end cross linking immunoprecipitation, seCLIP，一种增强型iCLIP方法）实验，旨在检测Rnmt cKO小鼠与对照小鼠的初始CD4 T细胞中LARP1的RNA结合位点。

实验整体设计如下：从小鼠淋巴结中磁分选（magnet sorted）获得初始CD4 T细胞，以此制备LARP1 seCLIP文库。具体实验流程为：先利用紫外线（UV light）将蛋白质与RNA交联，随后裂解细胞并对RNA进行部分酶切，通过免疫沉淀获取与LARP1交联的RNA，以此完成文库构建。本研究首先对比了三种不同的LARP1抗体在CD4 T细胞seCLIP实验中的应用效果，随后选定最优抗体，分别对Rnmt fl/fl（对照）与Rnmt fl/fl CD4cre+（Rnmt cKO）小鼠的CD4 T细胞制备文库。每个实验均设置LARP1抗体组、同型对照抗体（isotype control antibody）组及分子量匹配的输入对照（size matched input control）组。