Abstract
Frameshift mutations yield truncated proteins, leading to loss-of-function, genetic disorders or even death. Reverse mutations, which restore the wild-type phenotype of a mutant, was assumed to be far rarer than forward mutations. However, in this study, screening tests showed that the revertants of a frameshift mutation were detected more frequently than expected in E. coli. Sanger sequencing revealed that reverse mutations were caused not by random mutagenesis but by active targeted gene repair. Molecular studies suggested that it was the premature termination codons (PTCs) in nonsense mRNAs that signaled the repair of the frameshift mutation. Genome survey indicated that the genome sequence of a revertant is not more variable than that of a wild-type strain. Transcriptome profiling identified differentially expressed genes and pathways that were upregulated in frameshift or revertant which possibly involved in frameshift repair, include DNA replication, RNA surveillance, RNA editing, mismatch repair and homologous recombination. Introducing synthetic DNA or RNA oligonucleotides into the mutant increased the recovery rates as they promoted the frameshift repair. Based on these data, we hypothesized a molecular model for frameshift repair referred to as nonsense mRNA-mediated gene editing (NMGE): nonsense mRNAs were recognized by mRNA surveillance by PTCs signaling, edited by RNA editing and used to direct the repair of their defective coding gene through mismatch repair and homologous recombination. In addition, this mechanism also serve as a driving force for molecular evolution and the widespread presence of frameshift homologs within and across species is considered as evolutionary evidences preserved in nature.
Footnotes
Abbreviations used in this article:
- E. coli
- Escherichia coli
- BLA
- β-lactamase
- bla
- BLA gene
- HSC
- hidden stop codon
- PTC
- premature termination codon
- OE-PCR
- overlapping extension polymerase chain reaction
- SDS-PAGE
- sodium dodecyl sulfate polyacrylamide gel electrophoresis
- TCB
- tetracycline-containing broth
- TCP
- tetracycline-containing plate
- ACP
- ampicillin-containing plate
- ACB
- ampicillin-containing broth
- KCP
- kanamycin-containing plate
- KCB
- kanamycin-containing broth
- 2nd NGS
- the second-generation high-throughput sequencing
- DEG
- differentially expressed gene
- GO
- Gene Ontology
- KEGG
- Kyoto Encyclopedia of Genes and Genomes
- KOBAS
- KEGG Orthology Based Annotation System
- FPKM
- expected number of Fragments PerKilobase of transcript sequence per Millions base pairs sequenced
- SNP
- Single Nucleotide Polymorphism
- InDel
- Insertions/Deletion
- SV
- Structure Variation
- DSB
- double strand break