Identification of RISC-associated microRNAs and their targets during CD8⁺ T cell activation
MicroRNAs (miRNAs) are short (~22 nucleotide long) single-stranded noncoding RNAs that regulate gene expression post-transcriptionally in the RNA-induced silencing complex (RISC). miRNAs play an important role in immune cell function and affect many aspects of T cell immunity. Activation of naive T cells induces dramatic changes in the expression of miRNAs and RISC-associated proteins. We studied these changes in expression of miRNAs in CD8+ T cells using the OT-I transgenic T-cell receptor (TCR) mouse model, in which all T cells are CD8+ and respond to ovalbumin peptides. Upon in vitro activation, we saw dynamic changes in the expression of individual miRNAs, which were influenced by whether the T cells responded to high or low affinity peptides and whether they were differentiating to effector or memory cells. It was recently shown that in naive T cells, miRNAs are predominantly found in a low molecular weight (LMW) RISC composed of Argonaute (Ago)-proteins and miRNAs. Upon activation of T cells, biologically active miRNAs interacting with their target messenger RNAs (mRNAs) were shown to redistribute to a high molecular weight (HMW) RISC, which additionally contains RNA metabolism factors and Ago-interacting proteins such as GW182. We followed the development of HMW and LMW complexes in activated CD8⁺ T cells in order to determine their role and to identify the miRNAs and their targets present in both. We confirmed that GW182 protein was induced upon CD8⁺ T cell activation and associated with Ago-2, forming HMW complexes. To study the distribution of miRNAs between HMW and LMW RISC, we undertook small RNA sequencing of the associated miRNAs. From these data we identified specific miRNAs that were enriched in HMW RISC in activated CD8⁺ T cells. We also found that miRNA abundance did not always reflect its association with HMW RISC. Lastly, to discover miRNA targets, we used a novel method called cross-linking, ligation and sequencing of hybrids (CLASH), which directly identifies miRNAs and their targets by immunoprecipitation of RISC and RNA sequencing. From these data we found potential novel targets for key miRNAs in CD8⁺ T cells. Expanding our knowledge of the role of miRNAs in T cell activation beyond observations of miRNA expression changes, by focusing on biologically active miRNAs and their targets in HMW RISC will deepen our understanding of the mechanism of action of miRNAs as well as the signalling pathways surrounding T cell activation.