Analysis of High Molecular Weight RNA-induced silencing complex (HMW-RISC) in CD8+ T cells identifies miR-7 as a modulator of T cell activation

To identify the miRNAs expected to be potent in suppressing targets, we followed HMW RISC formation upon activation of CD8+ T cells. We show that while most miRNAs distribute between HMW and LMW RISC in activated T cells, several miRNAs were dominant in one complex over the other. Among these, miR-7 is enriched in HMW RISC and inhibition of miR-7 upon T cell activation leads to increased production of IL-2 and expression of IL-2-regulated proteins including the α-subunit of the IL-2 receptor, CD25; transferrin receptor, CD71; and amino acid transporter, CD98, which are direct miR-7 targets. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signalling and the metabolic processes regulated by IL-2 and thus modulates T cell activation. RNA was isolated from Ago2-IP samples, as well as the IP input and unbound samples. The samples were resuspended in QIAzol lysis reagent and RNA was extracted using the miRNeasy kit (Qiagen) and eluted in 100µl dH2O. The RNA was then ethanol precipitated by adding 2.5 x volume 100% ethanol, 30µl sodium acetate and 1µl GlycoBlue Coprecipitant (Thermo Scientific) to the samples which were then incubated overnight at -20oC. The following day, the samples were centrifuged at full speed for 30 min, then washed twice with 70% ethanol. The pellets were air-dried on ice then resuspended in 10-15µl dH2O and quantified with Qubit 3.0 fluorometer (Thermo Scientific) using the Qubit RNA HS Assay kit (Thermo Scientific). Small RNA libraries were prepared using the CleanTagTM Small RNA Library Preparation kit (Trilink) using manufacturer’s instructions and 21 cycles. The libraries were size selected to include 145-160 bps and the input, IP-unbound and IP-bound libraries were pooled at a 1:1:2 ratio. The samples were sequenced using NovaSeq 50bp paired end sequencing at Edinburgh Genomics.