Ribosome Profiling in the Model Diatom Thalassiosira pseudonana

Monica Pichler, Andreas Meindl, Markus Romberger, Annemarie Eckes-Shephard, Carl Fredrik Nyberg-Brodda, Claudia Buhigas, Sergio Llaneza-Lago, Gerhard Lehmann, Amanda Hopes, Gunter Meister, Jan Medenbach, Thomas Mock

Research output: Contribution to journalArticlepeer-review

Abstract

Diatoms are an important group of eukaryotic microalgae, which play key roles in marine biochemical cycling and possess significant biotechnological potential. Despite the importance of diatoms, their regulatory mechanisms of protein synthesis at the translational level remain largely unexplored. Here, we describe the detailed development of a ribosome profiling protocol to study translation in the model diatom Thalassiosira pseudonana, which can easily be adopted for other diatom species. To isolate and sequence ribosome-protected mRNA, total RNA was digested, and the ribosome-protected fragments were obtained by a combination of sucrose-cushion ultracentrifugation and polyacrylamide gel electrophoresis for size selection. To minimize rRNA contamination, a subtractive hybridization step using biotinylated oligos was employed. Subsequently, fragments were converted into sequencing libraries, enabling the global quantification and analysis of changes in protein synthesis in diatoms. The development of this novel ribosome profiling protocol represents a major expansion of the molecular toolbox available for diatoms and therefore has the potential to advance our understanding of the translational regulation in this important group of phytoplankton.

Original languageEnglish
Article numbere843
JournalCurrent Protocols
Volume3
Issue number7
DOIs
Publication statusPublished - 13 Jul 2023

Keywords

  • diatoms
  • high-throughput sequencing
  • ribosome profiling
  • RNA
  • Thalassiosira pseudonana
  • translation

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