Validation of the OR103-1/5 intergenic sequence as a molecular tool to promote bi-cistronic translation in zebrafish

Abstract

Sensory neuron in the olfactory system, typically expresses only a single olfactory receptor (OR) gene from a large and diverse genomic repertoire. In zebrafish, a violation of this ‘one neuron – one receptor rule’ has been identified with the systematic coexpression of two linked ORs. In previous studies we have provided evidence that coexpression of OR103- 1 and OR103-5 genes results from a failure to terminate transcription at the end of the upstream OR103-1 gene. We hypothesized that this unusual situation may lead to cotranslation of both OR genes by the same OSN through the presence of an internal ribosome entry site (IRES) within the OR103-1/5 intergenic region. The hypothesis was tested in vitro and in vivo by using dual-reporter constructs that contain renilla and firefly luciferase or two fluorescent reporter genes linked by the 1.4 kb intergenic region. Strong firefly and renilla luciferase activity in transfected HeLa cells suggests that the intergenic sequence may contain IRES activity in addition to cryptic promoter activity in the heterologous system. In transient transgenic zebrafish embryos injected with dual-reporter constructs a high correlation of GFP and mCherry fluorescence was observed in individual cells. Reporter gene coexpression was not restricted to olfactory tissue and was observed in other tissues when appropriate promoters were used. Colabeling was severly reduced when the reporter genes resided on different plasmids or when a polyadenylation signal was placed downstream of the first reporter. Substitution of the intergenic region with a different promoter equally abolished colabeling. In a final experiment we tried to coexpress OR proteins and fluorescent markers with the help of the bi-cistronic expression system. Forced overexpression of an OR gene in OSNs resulted in convergence of transgenic axons within the olfactory bulb. Under the influence of a strong enhancer, a subpopulation of axons converged onto novel glomeruli that were not present in control constructs that did not contain an OR sequence. In summary, the OR103-1 / 5 intergenic region promotes cotranslation of two linked gene coding sequences from a single transcript and can be used for successful coexpression of a marker gene and a gene of interest.