Project description:mTRAN proteins are are plant specific-components of the mitoribosome. To asses of loss-of-function mtran1 mtran2 double mutants show decreased translation rates, we performed ribosome footprinting coupled to RNA-seq (Ribo-seq).
Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
Project description:Charcot-Marie-Tooth (CMT) disease can be caused by mutations in Aminoacyl-tRNA-Synthetases, including G240R mutation in Glycyl-tRNA-Synthetase (GARS). Ribo-seq generates snapshots of translating ribosomes on mRNA and therefore allows analysis of ribosome pausing mRNA. Here we performed Ribo-seq on lysates of HEK293T cells overexpressing GARS, WT or G240R, to dissect mechanism of CMT linked with translation. We found that GARS G240R causes pausing of ribosomes with glycine codons in A-site. The effect is specific for 21 nt ribosome-protected fragments, produced by ribosomes with empty A-sites, suggestive of the deficit of charged Glycyl-tRNA in GARS G240R-CMT.
Project description:Ribo-seq was performed on cells treated with Tetracycline and Tylosin to to investigate the effect on ribosome pausing across the transcriptome of Escherichia coli MG1655.
Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of Saccharomyces cerevisiae with and without 10 minute glucose starvation to determine effects on translation.
Project description:We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC, which was achieved within 15 min from sample injection to fraction collection. Ribo Mega-SEC reproducibly shows translating ribosomes exist predominantly in polysome complexes in extracts isolated from human cell lines and mouse liver tissue, which alter in response to starvation. Ribo Mega-SEC provides a rapid, efficient, convenient and highly reproducible method for studying functional translation complexes and is easily combined with high-through put analysis such as proteomics and RNA-Seq, or with structural analysis using electron microscopy. We propose that Ribo Mega-SEC analysis is an accessible alternative to traditional polysome profiling using sucrose density gradients.
