A general framework to analyze potential roles of tDRs in mammalian protein synthesis.

tRNA-derived RNAs (tDRs) are a heterogeneous class of small non-coding RNAs that have been implicated in numerous biological processes including the regulation of mRNA translation. A subclass of tDRs called tRNA-derived stress-induced RNAs (tiRNAs) have been shown to participate in translational control under stress where specific tiRNAs repress protein synthesis. Here, we use a prototypical tiRNA (5'-tiRNA^Ala) that inhibits mRNA translation in vitro and in cells as a model to study potential roles of tDRs in translational control. Specifically, we propose to use commercially available and custom-made in vitro translation systems together with sensitive luciferase-based mRNA reporters as well as transfection studies to determine potential effects of a given tDR on various aspects of protein synthesis. We overview methods to probe the capacity of specific tDRs to target specific steps of mRNA translation initiation, the most regulated step in translational control. Using 5'-tiRNA^Ala as an example, we analyze its effects on the integrity of the m⁷GTP (cap)-bound eIF4F complex and phosphorylation of eIF2α, the key regulatory molecule of the Integrated Stress Response. Using transfection studies, we also monitor whether tDRs can promote formation of stress granules (SGs), RNA granules are often formed in response to global translation repression in live cells. This simple workflow offers fast, scalable, and reliable analyses of a potential involvement of specific tDRs in the modulation of protein synthesis and provides initial hints on molecular mechanisms that underline such mRNA translation regulation.