miRNA dynamics in T cell activation

Abstract

MiRNA repertoire of T cells undergoes extensive changes in response to activation. Beyond a global decrease in miRNA levels few hours after activation, some individual miRNAs are specifically up-regulated or down-regulated. We have assessed miRNA expression and post-transcriptional modification kinetics in human primary CD4+ T cells upon stimulation by T cell receptor (TCR) or with IFN I, using Next Generation Sequencing. Multiple miRNAs not related before with T cell activation profile have been identified as differentially expressed. Downregulated miRNAs presented higher 3’ uridylation, consistently with previous studies from our laboratory in mouse cells. Adenylation was overrepresented in upregulated miRNAs. In the midst of an adverse environment, with the likely presence of active miRNA degradation mechanisms, miRNAs multiplying their levels may use 3’ adenine as a protective signal. This potential strategy could open a new avenue to improve miRNA stability for therapy in T cells. Upon TCR stimulation, exoribonucleases Dis3L2 and Eri1 (previously described for its preferential degradation of uridylated substrates) upregulated their levels; together with terminal uridylyl transferases TUT4 and TUT7, that could be labelling their substrates. In the search of other relevant exoribonucleases in T cell context, we identified through mass spectrometry ISG20L2, a previously described 3’ to 5’ exoribonuclease, as an enzyme with a preferential interaction for uridylated miRNAs. We show ISG20L2 degrades miRNAs, with an efficiency influenced by nucleotides present at 3’ edge. In addition, we identify residues D267, D183 and E185 are involved in catalytic activity. In our search to find ISG20L2 substrates in T cells, so far miRNAs have not been found as the most affected population, at least in J77 non-activated cells. However, mRNA levels of various immunoregulatory molecules were largely affected, with upregulation of AHR, NKG2D, CTLA4, CD137, TIM3, PD-L1 and CD69. KO J77 clones were generated to study ISG20L2 function in T cells. IS20L2 had been described to be involved in ribosome biogenesis, but proliferation and cell survival of KO clones were not impaired. Remarkably, specific key molecules for T cell activation CD69, CD3ε, CD25, CTLA-4, PD-1 and IL2 were dysregulated.