OTTER, a new method for quantifying absolute amounts of tRNAs.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Accurate quantification of transfer RNA (tRNA) amounts is essential for understanding protein synthesis and proteome regulation.
• Existing high-throughput methods like RNA-Seq and microarrays have limitations in measuring absolute tRNA quantities and efficiencies.
• Cellular tRNA levels are more dynamic than previously assumed, necessitating precise measurement tools.

Purpose of the Study:

• To introduce and validate a novel method, Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), for quantifying absolute amounts of individual tRNA isoacceptors.
• To assess the dynamic range and physiological relevance of tRNA abundance variations in yeast.
• To compare the performance of OTTER with existing methods for tRNA quantification.

Main Methods:

• Development of the OTTER method utilizing fluorescence-labeling via optimized reverse primer extension.
• Assessment of labeling efficiency using northern blotting.
• Quantification of 34 individual and 4 isoacceptor tRNA pairs in Saccharomyces cerevisiae.

Main Results:

• OTTER successfully quantified absolute amounts of nuclear-encoded isoacceptor tRNAs in yeast, with levels ranging from 0.030 to 0.73 pmol/µg RNA.
• TRNA amounts showed significant alterations at the isoacceptor level in response to physiological growth conditions.
• OTTER data demonstrated poor correlation with RNA-Seq and marginal correlation with microarrays, but good agreement with 2D-gel analysis.

Conclusions:

• OTTER is a robust and suitable method for precise quantification of absolute tRNA amounts at the isoacceptor level.
• The method overcomes limitations of previous techniques, offering higher accuracy and resolution.
• Findings highlight the dynamic nature of tRNA populations and their importance in cellular regulation.