Utilizing high resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas
Ribosome profiling is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii for generating high-resolution data covering more than 10,000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5 prime- and 3 prime- sections of transcripts and features of eukaryotic translation termination, which are fundamentally different for chloroplast translation. In chloroplasts, a heterogenous ribosome protected fragment size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulator step during PsbA/D1 synthesis. Further analyses of ribosome protected fragment distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light harvesting complex proteins, LHCs. By providing high-quality data sets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim at making ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation of a gene of interest, to assay genome annotation and to compare translation of alternative transcripts.