Engineered transfer RNAs for suppression of premature termination codons
Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).
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Overall design. 16 ribosome footprinting samples are included. HEK-293 cells transiently transfected with 5 types of ACE-tRNA and a control plasmid (puc57GG) for 48 h with 2 biological replicates. G418-treated HEK-293 cells are also included with 2 biological replicates. Ribosome footprint data analysis. Data files for each barcoded sample (minus adaptor sequence at 3’ end) were first mapped to four ribosomal RNA sequences (RNA5S1;NR_023363, RNA5-8S;NR_003285, RNA18S;NR_003286, and RNA28S;NR_003287) using HISAT 2.0.3 to eliminate rRNA contaminant reads. The remaining reads were aligned to the sense stands of the longest transcript variant of each human gene (UCSC RefSeq GRCh38). Transcripts with 3’UTR length of at least 75-nt were used for subsequence analysis. A maximum of two mismatches at the 5’end of reads was allowed. All multi-mapped reads were discarded. Fragment reads with lengths between 26 to 34-nt were defined as ribosome footprints and used for analysis. The 5’ end nucleotide from each footprint was annotated and mapped on each transcript. Position of the ribosome A-site occupying the 16th-18th nucleotides of each footprint was used to infer the position of the ribosome on each transcript. Data files. Ribosome footprints mapped on the 3'UTRs are collected as a SAM file. The reference sequences (the longest transcript variant of each human gene from the UCSC RefSeq GRCh38) are collected as a FASTA file. The total count of ribosome footprints mapped on the transcriptome is shown in a tabular file.