Pervasive translation of the downstream ORF from bicistronic mRNAs by human cells: impact of the upstream ORF codon usage and splicing

Abstract

AbstractIn this work, we present a straightforward model to study gene expression regulation from virus-like bicistronic mRNAs in human cells, composed by a shortshbleupstream ORF and a reporteregfpdownstream ORF. We have engineered thirteen synonymous versions of theshbleORF to explore a large parameter space in compositional and folding features, as well as differences in propensity to undergo splicing. Our experimental model focuses on the control and modulation ofshbletranslation elongation and their effects on downstream translation: i) regarding translation initiation, all constructs share identical 5’UTR, ribosomal binding site andshblestart codon context; and ii) regardingegfptranslation, all constructs share identicalshblestop codon context, spacer betweenshbleandegfpand the fullegfpsequence. Our results show first that the human translation machinery can translate the downstream ORF in bicistronic mRNAs, independently of the characteristics of the upstream ORF. Even if GFP protein levels are hundreds of times lower than SHBLE protein levels, our results challenge the textbook interpretation positing that the eukaryotic translation machinery does not handle the downstream ORF in bicistronic transcripts. Second, we show that synonymous recoding of the upstream ORF determines its own translation efficiency, can uncover cryptic splice signals, and largely conditions the probability of translation from the downstream ORF. Our results are consistent with a leaky scanning mechanism at play for downstream ORF translation from bicistronic mRNAs in human cells.