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

Area of Science:

• Molecular Biology
• RNA Biology
• Yeast Genetics

Background:

• Noncoding RNAs, specifically tRNA-derived fragments (tDRs), are increasingly recognized as crucial regulators of gene expression.
• tDRs play a significant role in modulating protein biosynthesis, particularly under conditions of cellular stress.
• Quantitative methods for assessing in vivo tDR-ribosome interactions in yeast have been limited.

Purpose of the Study:

• To develop and validate a novel technique, tDR-quant, for the in vivo quantification of tDR-ribosome associations.
• To investigate the stress- and dose-dependency of tDR interactions with ribosomal subunits in Saccharomyces cerevisiae.
• To elucidate the functional impact of tDR-ribosome interactions on protein synthesis.

Main Methods:

• Development of tDR-quant: electroporation of radiolabeled tDRs into yeast spheroplasts.
• Application of polysome profiling and radioactivity detection to quantify tDR-ribosome binding.
• Validation using Northern blot and quantitative real-time PCR (qRT-PCR), alongside in vivo translation assays.

Main Results:

• 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.
• Elevated tDR levels inhibit protein synthesis without significantly altering overall polysome profiles.
• tDR-ribosome association patterns are stress-specific and do not directly correlate with tDR abundance, indicating selective binding.

Conclusions:

• tDR-quant is a reliable method for quantifying in vivo tDR-ribosome interactions in yeast.
• tDRs dynamically regulate translation by interacting with distinct ribosomal components in response to cellular stress.
• These findings highlight tDRs as key regulatory elements fine-tuning protein synthesis during stress responses in yeast.