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. 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.