Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE137567 | GEO | 2021/08/19
REPOSITORIES: GEO
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Project description:Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A
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Project description:Missense mutations account for nearly 50% of pathogenic mutations in human genetic diseases, most lack effective treatments. Gene therapies, CRISPR-based gene editing, and RNA therapies including transfer RNA (tRNA) modalities are common strategies for potential treatments of genetic diseases. However, reported tRNA therapies are for nonsense mutations, how tRNAs can be engineered to correct missense mutations have not been explored. Here, we describe missense correcting tRNAs (mc-tRNAs) as a potential therapeutic modality for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs that are charged with one amino acid and read codons of another amino acid in translation in human cells. We first developed a series of fluorescence protein (FP)-based reporters that indicate successful correction of missense mutations via restoration of fluorescence signals. We engineered mc-tRNAs that effectively corrected Serine and Arginine missense mutations in the reporters and confirmed the amino acid substitution by protein mass spectrometry and mc-tRNA expression by tRNA sequencing. We examined the transcriptome response to the expression of mc-tRNAs and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an Arg-tRNAGln(CUG) mc-tRNA to rescue the autolytic activity of a pathogenic CAPN3 Arg-to-Gln mutant involved in limb-girdle muscular dystrophy type 2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.
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Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
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Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
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