S2 cell culture, transfection, and luciferase reporter assays

Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) containing 10% FBS and Mycoplasma Prevention Reagent (TransGen FM501) without CO₂ to reach 2 × 10⁶ to 4 × 10⁶ cells/mL. Transfection was performed in ∼5 × 10⁵ cells per well using transfection reagent according to the manufacturer's instructions (JetPRIME).

We used Dual-Luciferase Reporter Assay System (Promega) by cloning three tandem repeats of an endogenous target site (a perfect 7-mer match to the nucleotides 2–8 of 5′-tsRNA + upstream 15 bp + downstream 8 bp; Supplemental Table S5) into the multiple cloning site (XholI/NotI) of psiCHECK-2. For 5′-tsRNA^Glu, 5′-tsRNA^Gly, and 5′-tsRNA^Pro, the target sites were placed in the 3′ UTR of Renilla; for 5′-tsRNA^Asp, the target sites were incorporated before the stop codon of Renilla. To contrast with the endogenous target sites, the mutated sites were created by scrambling the 7-mer seed match (Supplemental Table S5).

To examine the tsRNA actions and specificity, we performed co-transfection to quantify the effects of overexpression and sponge of tsRNAs on Renilla expression. For overexpression, cells were bathed with synthetic single-stranded RNA mimics of each 5′-tsRNA (Supplemental Table S3; Azenta) at a final concentration of 20 nM, along with the pAWH vector expressing the corresponding full-length tRNA (Supplemental Table S4). After transfection for 48 h, the luciferase activities of Firefly and Renilla were measured according to the manufacturer's instructions (Promega).