Abstract
Ribosomal profiling has revealed the translation of thousands of sequences outside of annotated protein-coding genes, including small Open Reading Frames of less than 100 codons, and the translational regulation of many genes. Here we have improved Poly-Ribo-Seq and applied it to Drosophila melanogaster embryos in order to extend the catalogue of in-vivo translated small ORFs, and to reveal the translational regulation of both small and canonical ORFs from mRNA across embryogenesis. We obtain highly correlated samples across five embryonic stages, with some 500 million putative ribosomal footprints mapped to mRNAs, and compared them to existing Riboseq and proteomics data. Our analysis reveals, for the first time in Drosophila, footprints mapping to codons as the unequivocal hallmark of productive translation, and we propose a simple binomial probability metric to ascertain translation probability. However, our results also reveal reproducible ribosomal binding apparently not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5’ mRNA Leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal to zygotic transition. We suggest that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding, and to incomplete ribosomal scanning of ORFs outside periods of productive translation. Finally, we show that the main function of upstream short ORFs is to buffer the translation of canonical ORFs, and that in general small ORFs in mRNAs display Poly-Ribo-Seq and bioinformatics markers compatible with an evolutionary transitory state towards full coding function.
