Translation initiation is emerging as one of the key mechanisms in regulation of gene expression. Translation is usually initiated from the first 5’ ATG codon with the certain flanking sequence, e.g. Kozak sequence. In the presence/occurrence of nonsense mutation near 5’ region in ORF, translation is sometimes reinitiated from a downstream inframe ATG codon, producing mutant protein lacking an N-terminal region. Such N-truncated protein has been reported as a responsible genetic factor for several human diseases. Some shortened proteins act as a hypomorphic protein and others act as a dominant negative protein. However, it has been known at all neither what extent translation reinitiation universally occurs nor how we may generally assess even if it may exist.
Here we show the concrete case of translation initiation in vivo and how to validate the event in vitro by using a dual-tagged expression vector. In the process of CRISPR-Cas9 genome editing, we have succeeded in introducing several out-of-frame indel mutations biallelically to knockout the endogenous Gli3 gene. Unexpectedly, almost full length of the GLI3 protein was expressed in the homozygous mutants in vivo. The dual-tagged vector revealed that the expressed mutant GLI3 proteins lacked N-terminal residues but had intact C-terminal.
Thus, translation reinitiation indeed occurred in vivo. The in vivo assessment system with the dual-tagged vector is practically applicable for any genes, e.g., for the diagnosis of human diseases as well as for designing genome-editing vectors.
Moreover, translation reinitiation has been also found without mutations; e.g., upstream open reading frames (uORFs) regulate gene expression in response to environmental conditions. Thus, the time has come to investigate if in general translation reinitiation has a critical regulatory role of gene expression as a new paradigm in the central dogma.