tDR-quant: a reliable electroporation-based approach for quantifying tRNA-derived fragments binding to ribosomes.

Ribosome-associated noncoding RNAs, particularly tRNA-derived fragments (tDRs), have emerged as key regulators of translation, especially under stress conditions. In Saccharomyces cerevisiae, tDRs interact with small ribosomal subunits to modulate protein biosynthesis, yet methods to quantitatively assess these interactions have been lacking. Here, we present tDR-quant, a robust technique for in vivo quantification of tDR/ribosome associations using electroporation of radiolabeled tDRs into yeast spheroplasts, followed by polysome profiling and radioactivity detection. We show that tDR interactions with ribosomes are stress- and dose-dependent, primarily associating with the 40S subunit but also with 60S, monosomes, and polysomes under specific conditions. Translation assays revealed that increased tDR levels inhibit protein synthesis without altering polysome profiles. Northern blot and quantitative real-time PCR (qRT-PCR) validated tDR-quant results, confirming its reliability. Stress-specific association patterns suggest that tDRs dynamically regulate translation by interacting with different ribosomal components in response to environmental cues. Importantly, these interactions do not correlate directly with tDR abundance, indicating selective ribosome binding. This study provides the first comprehensive method to quantify tDR-ribosome interactions in vivo and demonstrates that tDRs act as regulatory elements fine-tuning translation during cellular stress in yeast.