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.

The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

Evolution of Hox post-transcriptional regulation by alternative polyadenylation and microRNA modulation within 12 Drosophila genomes.

Hox genes encode a family of transcriptional regulators that operate differential developmental programs along the anteroposterior axis of bilateral animals. Regulatory changes affecting Hox gene expression are believed to have been crucial for the evolution of animal body plans. In Drosophila melanogaster, Hox expression is post-transcriptionally regulated by microRNAs (miRNAs) acting on target sites located in the 3' untranslated regions (3'UTRs) of Hox mRNAs. Notably, recent work has shown that during D. melanogaster development Hox genes produce mRNAs with variable 3'UTRs (short and long forms) in different sets of tissues as a result of alternative polyadenylation; importantly, Hox short and long 3'UTRs contain very different target sites for miRNAs. Here, we use a computational approach to explore the evolution of Hox 3'UTRs treated with especial regard to miRNA regulation. Our work is focused on the 12 Drosophila species for which genomic sequences are available and shows, first, that alternative polyadenylation of Hox transcripts is a feature shared by all drosophilids tested in the study. Second, that the regulatory impact of miRNAs is evolving very fast within the Drosophila group. Third, that in contrast to the low degree of primary sequence conservation, Hox 3'UTR regions within the group show very similar RNA topology indicating that RNA structure is under strong selective pressure. Finally, we also demonstrate that Hox alternative polyadenylation can remodel the control regions seen by miRNAs by at least two mechanisms: via adding new cis-regulatory sequences-in the form of miRNA target sites-to short 3'UTR forms as well as by modifying the regulatory impact of miRNA target sites in short 3'UTR forms through changes in RNA secondary structure caused by the use of distal polyadenylation signals.

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".

Plant-microbe symbioses: new insights into common roots.

Arbuscular mycorrhiza (AM), a type of plant-fungal endosymbiosis, and nodulation, a bacterial-plant endosymbiosis, are the most ubiquitous symbioses on earth. Recent findings have established part of a shared genetic basis underlying these interactions. Here, we approach root endosymbioses through the lens of the homology and modularity concepts aiming at further clarifying the proximate and ultimate causes for the establishment of these biological systems. We review the genetics that underlie interspecific signaling and its concomitant shift in genetic programs for either partner. Also, through the comparative analysis of genetic modules shared by AM and nodulation symbioses, we identify fundamental nodes in these networks, suggesting the elemental steps that may have permitted symbiotic adaptation. Here, we show that this approach, allied to recent technical advances in the study of genetic systems architecture, can provide clear testable hypotheses for the advancement of our understanding on the evolution and development of symbiotic systems.

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.

Pervasive Behavioral Effects of MicroRNA Regulation in Drosophila.

The effects of microRNA (miRNA) regulation on the genetic programs underlying behavior remain largely unexplored. Despite this, recent work in Drosophila shows that mutation of a single miRNA locus (miR-iab4/iab8) affects the capacity of the larva to correct its orientation if turned upside down (self-righting, SR), suggesting that other miRNAs might also be involved in behavioral control. Here we explore this possibility, studying early larval SR behavior in a collection of 81 Drosophila miRNA mutants covering almost the entire miRNA complement of the late embryo. Unexpectedly, we observe that >40% of all miRNAs tested significantly affect SR time, revealing pervasive behavioral effects of miRNA regulation in the early larva. Detailed analyses of those miRNAs affecting SR behavior (SR-miRNAs) show that individual miRNAs can affect movement in different ways, suggesting that specific molecular and cellular elements are affected by individual miRNA mutations. Furthermore, gene expression analysis shows that the Hox gene Abdominal-B (Abd-B) represents one of the targets deregulated by several SR-miRNAs. Our work thus reveals pervasive effects of miRNA regulation on a complex innate behavior in Drosophila and suggests that miRNAs may be core components of the genetic programs underlying behavioral control in other animals too.