Classification and function of small open reading frames.

Small open reading frames (smORFs) of 100 codons or fewer are usually - if arbitrarily - excluded from proteome annotations. Despite this, the genomes of many metazoans, including humans, contain millions of smORFs, some of which fulfil key physiological functions. Recently, the transcriptome of Drosophila melanogaster was shown to contain thousands of smORFs of different classes that actively undergo translation, which produces peptides of mostly unknown function. Here, we present a comprehensive analysis of smORFs in flies, mice and humans. We propose the existence of several functional classes of smORFs, ranging from inert DNA sequences to transcribed and translated cis-regulators of translation and peptides with a propensity to function as regulators of membrane-associated proteins, or as components of ancient protein complexes in the cytoplasm. We suggest that the different smORF classes could represent steps in gene, peptide and protein evolution. Our analysis introduces a distinction between different peptide-coding classes of smORFs in animal genomes, and highlights the role of model organisms for the study of small peptide biology in the context of development, physiology and human disease.

Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution.

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. We compared the expression and function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution.

Functions of long non-coding RNAs in human disease and their conservation in Drosophila development.

Genomic analysis has found that the transcriptome in both humans and Drosophila melanogaster features large numbers of long non-coding RNA transcripts (lncRNAs). This recently discovered class of RNAs regulates gene expression in diverse ways and has been involved in a large variety of important biological functions. Importantly, an increasing number of lncRNAs have also been associated with a range of human diseases, including cancer. Comparative analyses of their functions among these organisms suggest that some of their modes of action appear to be conserved. This highlights the importance of model organisms such as Drosophila, which shares many gene regulatory networks with humans, in understanding lncRNA function and its possible impact in human health. This review discusses some known functions and mechanisms of action of lncRNAs and their implication in human diseases, together with their functional conservation and relevance in Drosophila development.

Discovery and characterization of smORF-encoded bioactive polypeptides.

Analysis of genomes, transcriptomes and proteomes reveals the existence of hundreds to thousands of translated, yet non-annotated, short open reading frames (small ORFs or smORFs). The discovery of smORFs and their protein products, smORF-encoded polypeptides (SEPs), points to a fundamental gap in our knowledge of protein-coding genes. Various studies have identified central roles for smORFs in metabolism, apoptosis and development. The discovery of these bioactive SEPs emphasizes the functional potential of this unexplored class of biomolecules. Here, we provide an overview of this emerging field and highlight the opportunities for chemical biology to answer fundamental questions about these novel genes. Such studies will provide new insights into the protein-coding potential of genomes and identify functional genes with roles in biology and disease.

Ribosomal profiling adds new coding sequences to the proteome.

Next generation sequencing (NGS) has enabled an in-depth look into genes, transcripts and their translation at the genomic scale. The application of NGS sequencing of ribosome footprints (Ribo-Seq) reveals translation with single nucleotide (nt) resolution, through the deep sequencing of ribosome-bound fragments (RBFs). Some results of Ribo-Seq challenge our understanding of the protein-coding potential of the genome. Earlier bioinformatic approaches had shown the presence of hundreds of thousands of putative small ORFs (smORFs) in eukaryotic genomes, but they had been largely ignored due to their large numbers and difficulty in determining their translation and function. Ribo-Seq has revealed that hundreds of putative smORFs within previously assumed long non-coding RNAs (lncRNAs) and UTRs of canonical mRNAs are associated with ribosomes, appearing to be translated. Here we review some of the approaches used to define translation within Ribo-Seq experiments and the challenges in defining translation of these novel smORFs in lncRNAs and UTRs. We also look at some of the bioinformatic and biochemical approaches used to independently corroborate these exciting new findings and elucidate real translation events.

Tarsal-less peptides control Notch signalling through the Shavenbaby transcription factor.

The formation of signalling boundaries is one of the strategies employed by the Notch (N) pathway to give rise to two distinct signalling populations of cells. Unravelling the mechanisms involved in the regulation of these signalling boundaries is essential to understanding the role of N during development and diseases. The function of N in the segmentation of the Drosophila leg provides a good system to pursue these mechanisms at the molecular level. Transcriptional and post-transcriptional regulation of the N ligands, Serrate (Ser) and Delta (Dl) generates a signalling boundary that allows the directional activation of N in the distalmost part of the segment, the presumptive joint. A negative feedback loop between odd-skipped-related genes and the N pathway maintains this signalling boundary throughout development in the true joints. However, the mechanisms controlling N signalling boundaries in the tarsal joints are unknown. Here we show that the non-canonical tarsal-less (tal) gene (also known as pri), which encodes for four small related peptides, is expressed in the N-activated region and required for joint development in the tarsi during pupal development. This function of tal is both temporally and functionally separate from the tal-mediated tarsal intercalation during mid-third instar that we reported previously. In the pupal function described here, N signalling activates tal expression and reciprocally Tal peptides feedback on N by repressing the transcription of Dl in the tarsal joints. This Tal-induced repression of Dl is mediated by the post-transcriptional activation of the Shavenbaby transcription factor, in a similar manner as it has been recently described in the embryo. Thus, a negative feedback loop involving Tal regulates the formation and maintenance of a Dl+/Dl- boundary in the tarsal segments highlighting an ancient mechanism for the regulation of N signalling based on the action of small cell signalling peptides.

Control of Distal-less expression in the Drosophila appendages by functional 3' enhancers.

The expression of the Hox gene Distal-less (Dll) directs the development of appendages in a wide variety of animals. In Drosophila, its expression is subjected to a complex developmental control. In the present work we have studied a 17kb genomic region in the Dll locus which lies downstream of the coding sequence and found control elements of primary functional importance for the expression of Dll in the leg and in other tissues. Of particular interest is a control element, which we have called LP, which drives expression of Dll in the leg primordium from early embryonic development, and whose deletion causes severe truncation and malformation of the adult leg. This is the first Dll enhancer for which, in addition to the ability to drive expression of a reporter, a role can be demonstrated in the expression of the endogenous Dll gene and in the development of the leg. In addition, our results suggest that some enhancers, contrary to the widely accepted notion, may require a specific 5' or 3' position with respect to the transcribed region.

Translation and natural selection of micropeptides from long non-canonical RNAs.

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".

Pegasus, a small extracellular peptide enhancing short-range diffusion of Wingless.

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.

Peptides encoded by short ORFs control development and define a new eukaryotic gene family.

Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal), a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs), which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.

Developmental regulation of canonical and small ORF translation from mRNAs.

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.

The 11-aminoacid long Tarsal-less peptides trigger a cell signal in Drosophila leg development.

The polycistronic and non-canonical gene tarsal-less encodes several short peptides 11 to 32 aminoacids long. tarsal-less is required for embryonic and imaginal development in Drosophila, but the molecular and cellular bases of its function are not known. Here we show that tarsal-less function triggers a cell signal. This signal has a range of 2-3 cells in Drosophila legs and may be provided directly by the Tarsal-less peptides. During leg development, this Tarsal-less signal implements the patterning activity of a tarsal boundary and regulates the transcription of several genes in a specific manner. Thus tarsal-less is necessary for the intercalation of the tarsal segments two to four and for the activation of the homeobox gene apterous, the Zinc-finger gene rotund and the bHLH-PAS gene spineless, and for the repression of the homeobox gene Bar and the putative transcription factor dacshund. These regulatory effects complement the known genetic scenario required for distal leg development and explain the requirements for tarsal-less in this process.

Parallels between the proximal-distal development of vertebrate and arthropod appendages: homology without an ancestor?

Evolutionary studies suggest that the limbs of vertebrates and the appendages of arthropods do not share a common origin. However, recent genetic studies show new similarities in their developmental programmes. These similarities might be caused by the independent recruitment of homologous genes for similar functions or by the conservation of an ancestral proximal-distal development programme. This basic programme might have arisen in an ancestral outgrowth and been independently co-opted in vertebrate and arthropod appendages. It has subsequently diverged in both phyla to fine-pattern the limb and to control phylum-specific cellular events. We suggest that although vertebrate limbs and arthropod appendages are not strictly homologous structures they retain remnants of a common ancestral developmental programme.

Finding smORFs: getting closer.

Millions of small open reading frames exist in eukaryotes. We do not know how many, or which are translated, but bioinformatics is getting us closer to the answer. See related Research article: http://www.genomebiology.com/2015/16/1/179.

Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction.

Up to 80% of individuals with myotonic dystrophy type 1 (DM1) will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage of varying degrees. Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts, as revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in humans with DM1, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function, reduced heart tube diameters and reduced contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.

Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq.

Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The 'longer' smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The 'dwarf' smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5'-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.

Interplay between a Wnt-dependent organiser and the Notch segmentation clock regulates posterior development in Periplaneta americana.

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.

Activation of Drosophila hemocyte motility by the ecdysone hormone.

Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.