Utilizing high resolution ribosome profiling for the global investigation of gene expression inChlamydomonas

Abstract Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Here, we established an optimized and high resolution Ribo-seq protocol for the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas). Comparing different nuclease treatments for the extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq, provided deep insight into translational dynamics and post-transcriptional control of gene expression, thoroughly covering more than 10,000 different transcripts. Our high quality Ribo-seq protocol captures the 3-nucleotide movement of elongating ribosomes along nuclear and chloroplast transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5’- and 3’-sections of transcripts and features of eukaryotic translation termination. These offsets reveal drastic differences between the nature of cytosolic and chloroplast translation mechanisms. Chloroplast translation is further characterized by heterogenous RPF size distribution. We found that local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly revealing an uncharacterized regulator step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light harvesting complex proteins, LHCs. Moreover, our Ribo-seq data can be utilized to survey coding sequence annotations and the expression preference of alternatively spliced transcripts in Chlamydomonas. We made these features easily accessible for the research community by attaching our Ribo-seq data to the most recent Chlamydomonas reference genome.