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Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli
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OTTER, a new method for quantifying absolute amounts of tRNAs.

Akihisa Nagai1, Kohei Mori1, Yuma Shiomi1

  • 1Graduate School of Life Science, University of Hyogo, Ako-gun, Hyogo 678-1297, Japan.

RNA (New York, N.Y.)
|March 6, 2021
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Summary

Researchers developed a new method, Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), to accurately measure absolute amounts of transfer RNAs (tRNAs). This technique provides precise quantification of tRNA isoacceptors, crucial for understanding protein synthesis regulation.

Keywords:
absolute amountquantificationtRNA

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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.