2. Academic Validation
  3. Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts

  • Nat Commun. 2024 Aug 26;15(1):7061. doi: 10.1038/s41467-024-51258-0.
Kotaro Tomuro # 1 2,  Mari Mito # 1,  Hirotaka Toh 1,  Naohiro Kawamoto 1,  Takahito Miyake 3,  Siu Yu A Chow 4,  Masao Doi 3,  Yoshiho Ikeuchi 4 5 6,  Yuichi Shichino 7,  Shintaro Iwasaki 8 9
Affiliations

Affiliations

  • 1 RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, 351-0198, Japan.
  • 2 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan.
  • 3 Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyō-ku, Kyoto, 606-8501, Japan.
  • 4 Institute of Industrial Science, The University of Tokyo, Meguro-ku, Tokyo, 153-8505, Japan.
  • 5 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
  • 6 Institute for AI and Beyond, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
  • 7 RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, 351-0198, Japan. yuichi.shichino@riken.jp.
  • 8 RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, 351-0198, Japan. shintaro.iwasaki@riken.jp.
  • 9 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan. shintaro.iwasaki@riken.jp.
  • # Contributed equally.
PMID: 39187487 DOI: 10.1038/s41467-024-51258-0
Abstract

Ribosome profiling, which is based on deep Sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.

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