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Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Author

Listed:
  • Kotaro Tomuro

    (RIKEN Cluster for Pioneering Research
    The University of Tokyo)

  • Mari Mito

    (RIKEN Cluster for Pioneering Research)

  • Hirotaka Toh

    (RIKEN Cluster for Pioneering Research)

  • Naohiro Kawamoto

    (RIKEN Cluster for Pioneering Research)

  • Takahito Miyake

    (Kyoto University, Sakyō-ku)

  • Siu Yu A. Chow

    (The University of Tokyo)

  • Masao Doi

    (Kyoto University, Sakyō-ku)

  • Yoshiho Ikeuchi

    (The University of Tokyo
    The University of Tokyo
    The University of Tokyo)

  • Yuichi Shichino

    (RIKEN Cluster for Pioneering Research)

  • Shintaro Iwasaki

    (RIKEN Cluster for Pioneering Research
    The University of Tokyo)

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.

Suggested Citation

  • Kotaro Tomuro & Mari Mito & Hirotaka Toh & Naohiro Kawamoto & Takahito Miyake & Siu Yu A. Chow & Masao Doi & Yoshiho Ikeuchi & Yuichi Shichino & Shintaro Iwasaki, 2024. "Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-51258-0
    DOI: 10.1038/s41467-024-51258-0
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    References listed on IDEAS

    as
    1. Shiho Makino & Tomoko Kawamata & Shintaro Iwasaki & Yoshinori Ohsumi, 2021. "Selectivity of mRNA degradation by autophagy in yeast," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    2. Shintaro Iwasaki & Stephen N. Floor & Nicholas T. Ingolia, 2016. "Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor," Nature, Nature, vol. 534(7608), pages 558-561, June.
    3. Kazuhiro Kashiwagi & Yuichi Shichino & Tatsuya Osaki & Ayako Sakamoto & Madoka Nishimoto & Mari Takahashi & Mari Mito & Friedemann Weber & Yoshiho Ikeuchi & Shintaro Iwasaki & Takuhiro Ito, 2021. "eIF2B-capturing viral protein NSs suppresses the integrated stress response," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
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