Developmental regulation of canonical and small ORF translation from mRNAs

Pedro Patraquim,Julie Louise Aspden,Muhammad Ali Shahzad Mumtaz,Juan-Pablo Couso,José Ignacio Pueyo

doi:10.1186/s13059-020-02011-5

Pedro Patraquim, Julie Louise Aspden + Show 3 more

Open Access

https://doi.org/10.1186/s13059-020-02011-5

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Abstract

BackgroundRibosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. Here we present an improved version of Poly-Ribo-Seq and apply it to Drosophila melanogaster embryos to extend the catalog of in vivo translated small ORFs, and to reveal the translational regulation of both small and canonical ORFs from mRNAs across embryogenesis.ResultsWe obtain highly correlated samples across five embryonic stages, with nearly 500 million putative ribosomal footprints mapped to mRNAs, and compare them to existing Ribo-Seq and proteomic data. Our analysis reveals, for the first time in Drosophila, footprints mapping to codons in a phased pattern, the hallmark of productive translation. We propose a simple binomial probability metric to ascertain translation probability. 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.ConclusionsWe suggest that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding and to defective ribosomal scanning of ORFs outside periods of productive translation. Our results are compatible with the main function of upstream short ORFs being to buffer the translation of canonical canonical ORFs; and show that, in general, small ORFs in mRNAs display markers compatible with an evolutionary transitory state towards full coding function.

Highlights

Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes
The most thorough proteomics study to date covering the whole Drosophila melanogaster life-cycle has detected less than 40% of all unique canonical proteins [26]. This number is further reduced to 30% of annotated smORF polypeptides, while we have previously reported that 80% of canonical and small ORFs show clear Ribo-Seq evidence of translation in a single embryonic cell line [23]
Our results reveal reproducible ribosomal binding 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

Summary

Introduction

Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. During the first 2 h after egg laying (AEL), there is absence of transcription from the zygotic genome and the key developmental processes, such as establishment of the primary antero-posterior and dorso-ventral axes, are controlled purely through the translational regulation of maternal mRNA previously laid down in the egg [16, 17]. After this initial period, the embryo undergoes a “maternal to zygotic transition,” whereby the transcription and translation of the zygotic genome takes over the maternal products, a process found in nematodes, echinoderms, and vertebrates [13, 18,19,20]. The impact of translational regulation at the genomewide scale on the whole of Drosophila embryogenesis has not yet been revealed

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