Vincent Leon Gotsmann

• Translation is a ubiquitous, yet enigmatic process central to all cellular life on earth. Despite being a subject of research for many decades, its enormous complexity makes it difficult to study translation. The development of the Ribo-Seq technique, a combination of classical ribosome profiling and next generation sequencing, enabled scientists to get a closer view at translating ribosomes than ever before. This technique allows to investigate the topic with unprecedented detail and to generate deep insights into the mechanics and regulation of translation. In this study, a Ribo-Seq protocol for C. reinhardtii and a Python module for the analysis of Ribo-Seq data was presented. The data generated was analyzed translatome-wide and its quality was assessed regarding sequencing depth, the RPF’s frame preference and their length distribution. The data delivered deep insights into the mixotrophic translatome, clearly demonstrating the dominance of anabolism-related transcripts. Analysis of 5’-P-site offsets shed light on the mechanics of cytosolic translation revealing a clear tri-nucleotide periodicity of cytosolic RPFs. It was shown that ribosome profiles reproducibly reflect the dynamics of translation and can be utilized to refine genomic annotations. The appearance of an RNAi-related ribosome-binding protein (VIG1) among the 150 top-translated transcripts, together with the frequent observation of extreme RPF-coverage within initiation regions indicates that regulation of cytosolic translation in C. reinhardtii may be more complex than previously anticipated. Analysis of chloroplast RPFs indicated that chloroplasts evolved highly specific mechanisms to fine-tune translation and that translational activity in the chloroplast may be regulated on a high level by the titers or ribosomal proteins Rpl20 and PRPL7. An analysis of the translatome of a knock-down mutant of uS11c revealed four transcripts depending on the protein for translation as well as a list of transcripts putatively involved in the formation of palmelloid colonies. Additionally, a seRP approach suggested psaB and ftsH as targets of a transient co-translational interaction of cpSRP54 with the chloroplast ribosome and pointed towards the protein’s involvement in a putative co-translational protein import pathway into the chloroplast. Parts of this study have been published previously in a scientific Journal in the publication “Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas” (Gotsmann et al., 2024). Furthermore, the RPF coverage of genes in the C. reinhardtii genome, version 6.1 was published on the Phytozome platform (Goodstein et al., 2012) to enable the scientific community to compare existing genomic annotations against Ribo-Seq data.