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
