Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides but lacking canonical coding sequences. Apparently unable to produce peptides, lncRNA function seems to rely only on RNA expression, sequence and structure. Here, we exhaustively detect in-vivo translation of small open reading frames (small ORFs) within lncRNAs using Ribosomal profiling during Drosophila melanogaster embryogenesis. We show that around 30% of lncRNAs contain small ORFs engaged by ribosomes, leading to regulated translation of 100 to 300 micropeptides. We identify lncRNA features that favour translation, such as cistronicity, Kozak sequences, and conservation. For the latter, we develop a bioinformatics pipeline to detect small ORF homologues, and reveal evidence of natural selection favouring the conservation of micropeptide sequence and function across evolution. Our results expand the repertoire of lncRNA biochemical functions, and suggest that lncRNAs give rise to novel coding genes throughout evolution. Since most lncRNAs contain small ORFs with as yet unknown translation potential, we propose to rename them “long non-canonical RNAs”.

Summary: Sequential addition of segments in the posteriorly growing end of the embryo is a developmental mechanism common to many bilaterians. However, posterior growth and patterning in most animals also entails the establishment of a ‘posterior organiser’ that expresses the Caudal and Wnt proteins and has been proposed to be an ancestral feature of animal development. We have studied the functional relationships between the Wnt-driven organiser and the segmentation mechanisms in a basal insect, the cockroach Periplaneta americana. Here, posteriorly-expressed Wnt1 promotes caudal and Delta expression early in development to generate a growth zone from which segments will later bud off. caudal maintains the undifferentiated growth zone by dampening Delta expression, and hence Notch-mediated segmentation occurs just outside the caudal domain. In turn, Delta expression maintains Wnt1, maintaining this posterior gene network until all segments have formed. This feedback between caudal, Wnt and Notch-signalling in regulating growth and segmentation seems conserved in other arthropods, with some aspects found even in vertebrates. Thus our findings not only support an ancestral Wnt posterior organiser, but also impinge on the proposals for a common origin of segmentation in arthropods, annelids and vertebrates.

Background: 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. 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. Results: We 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. Conclusions: We 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.

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.

Recent advances in proteogenomic techniques and bioinformatic pipelines have permitted the detection of thousands of translated small Open Reading Frames (smORFs), which contain less than 100 codons, in eukaryotic genomes. Hundreds of these actively translated smORFs display conserved sequence, structure and evolutionary signatures indicating that the translated peptides could fulfil important biological roles. Despite their abundance, only tens of smORF genes have been fully characterised; these act mainly as regulators of canonical proteins involved in essential cellular processes. Importantly, some of these smORFs display conserved functions with their mutations being associated with pathogenesis. Thus, investigating smORF roles in Drosophila will not only expand our understanding of their functions but it may have an impact in human health. Here we describe the function of a novel and essential Drosophila smORF gene named purriato (prto). prto belongs to an ancient gene family whose members have expanded throughout the Protostomia clade. prto encodes a transmembrane peptide which is localized in endo-lysosomes and perinuclear and plasma membranes. prto is dynamically expressed in mesodermal tissues and imaginal discs. Targeted prto knockdown (KD) in these organs results in changes in nuclear morphology and endo-lysosomal distributions correlating with the loss of sarcomeric homeostasis in muscles and reduction of mitosis in wing discs. Consequently, prto KD mutants display severe reduction of motility, and shorter wings. Finally, our genetic interaction experiments show that prto function is closely associated to the CASA pathway, a conserved mechanism involved in turnover of mis-folded proteins and linked to muscle dystrophies and neurodegenerative diseases. Thus, this study shows the relevance of smORFs in regulating important cellular functions and supports the systematic characterisation of this class of genes to understand their functions and evolution.

Small Open Reading Frames (smORFs) of less than 100 codons remain mostly uncharacterised. About a thousand smORFs per genome encode peptides and microproteins about 70–80 aa long, often containing recognisable protein structures and markers of translation, and these are referred to as short Coding Sequences (sCDSs). The characterisation of individual sCDSs has provided examples of smORFs’ function and conservation, but we cannot infer the functionality of all other metazoan smORFs from these. sCDS function has been characterised at a genome-wide scale in yeast and bacteria, showing that hundreds can produce a phenotype, but attempts in metazoans have been less successful. Either most sCDSs are not functional, or classic experimental techniques do not work with smORFs due to their shortness. Here, we combine extensive proteomics with bioinformatics and genetics in order to detect and corroborate sCDS function in Drosophila. Our studies nearly double the number of sCDSs with detected peptides and microproteins and an experimentally corroborated function. Finally, we observe a correlation between proven sCDS protein function and bioinformatic markers such as conservation and GC content. Our results support that sCDSs peptides and microproteins act as membrane-related regulators of canonical proteins, regulators whose functions are best understood at the cellular level, and whose mutants produce little, if any, overt morphological phenotypes.