Ribosome profiling analysis of zebrafish embryos injected with the codon tag reporter library
Ontology highlight
ABSTRACT: We measured the effect of codons on mRNA stability in zebrafish embryos using reporter mRNAs. We showed that slow decoding of codons by the ribosome promoted mRNA degradation in zebrafish.
Project description:We measured the effect of codons on mRNA stability in zebrafish embryos using reporter mRNAs. We showed that slow decoding of codons by the ribosome promoted mRNA degradation in zebrafish.
Project description:We measured the effect of codons on mRNA stability in zebrafish embryos using reporter mRNAs. We showed that slow decoding of codons by the ribosome promoted mRNA degradation in zebrafish.
Project description:Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with both mRNA stability using a massive reporter library and expression levels using fluorescent reporters and analysis of endogenous gene expression in zebrafish embryos and/or human cells. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.
Project description:Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity. Ribosome profiling of wild-type and tRNA modification-deficient yeast and nematodes. Yeast samples were generated in various growth conditions (rich medium versus stress induced by treatment with diamide or rapamycin) and paired mRNA-Seq was performed on a subset of samples. Dataset contains three biological replicates for yeast samples and two biological replicates for nematode samples.
Project description:The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. In addition to a quantitative analysis of the protein products that are formed in dependence of the modified codons, the interpretation of these RNA nucleobase derivatives by the ribosomal decoding site was also determined. For each modification, the translated peptides from the bacterial and eukaryotic systems were purified and analyzed by mass spectrometry.
Project description:The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. In addition to a quantitative analysis of the protein products that are formed in dependence of the modified codons, the interpretation of these RNA nucleobase derivatives by the ribosomal decoding site was also determined. For each modification, the translated peptides from the bacterial and eukaryotic systems were purified and analyzed by mass spectrometry.
Project description:Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity.