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Optimized protocol for tRNA identification in the ribosomal complexes from human cell lines.

Tsuyoshi Udagawa1, Moeka Seki1, Toshifumi Inada1,2

  • 1Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 981-8567, Japan.

STAR Protocols
|June 30, 2021
PubMed
Summary

This study presents a protocol to identify transfer RNAs (tRNAs) within the 60S ribosome-nascent peptide complex. The method uses reverse transcription and quantitative PCR to estimate relative tRNA levels, aiding ribosome research.

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Cell separation/fractionationMolecular Biology

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Area of Science:

  • Molecular Biology
  • Ribosome Biology
  • RNA Biology

Background:

  • The 60S ribosome subunit plays a crucial role in protein synthesis.
  • Nuclear Export Mediator Factor (NEMF) is involved in nascent peptide modification.
  • Understanding tRNA dynamics in ribosome complexes is essential for deciphering translation regulation.

Purpose of the Study:

  • To develop a protocol for identifying transfer RNA (tRNA) species within the 60S ribosome-nascent peptide complex.
  • To facilitate the study of tRNA involvement in complexes associated with NEMF.
  • To provide a method for estimating relative tRNA levels in specific cellular contexts.

Main Methods:

  • Co-purification of the 60S ribosome-nascent peptide complex with Nuclear Export Mediator Factor (NEMF).
  • Application of reverse transcription to RNA extracted from the complex.
  • Utilizing quantitative Polymerase Chain Reaction (qPCR) for tRNA identification and quantification.

Main Results:

  • A protocol for tRNA identification in the 60S ribosome-NEMF complex was successfully established.
  • The method allows for the estimation of relative levels of different tRNA species.
  • The protocol is a convenient tool for researchers studying ribosome-nascent peptide interactions.

Conclusions:

  • The described protocol offers a practical approach for tRNA profiling in ribosome complexes.
  • This method can aid in understanding the role of specific tRNAs during nascent peptide processing.
  • Further research can build upon this protocol to investigate tRNA modifications and charging states.