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.DOI: http://dx.doi.org/10.7554/eLife.03528.001

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for a-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.[Keywords: Ubiquitin; K63-linked ubiquitin chains; splicing; Prp19 complex; Usp4] Supplemental material is available at http://www.genesdev.org.

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Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative premRNA splicing and small RNA pathways has remained elusive. Here we show that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, we show that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Importantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, we determined the nuclear RNA-binding profile of Ago-2, found it bound to several splicing target transcripts, and identified a G-rich RNA-binding site for Ago-2 that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression.

Our genomes contain the blueprint of what makes us human and many indications as to why we develop disease. Until the last 10 years, most studies had focussed on protein-coding genes, more specifically DNA sequences coding for proteins. However, this represents less than 5% of our genomes. The other 95% is referred to as the 'dark matter' of our genomes, our understanding of which is extremely limited. Part of this 'dark matter' includes regions that give rise to RNAs that do not code for proteins. A subset of these non-coding RNAs are long non-coding RNAs (lncRNAs), which in particular are beginning to be dissected and their importance to human health revealed. To improve our understanding and treatment of disease it is vital that we understand the molecular and cellular function of lncRNAs, and how their misregulation can contribute to disease. It is not yet clear what proportion of lncRNAs is actually functional; conservation during evolution is being used to understand the biological importance of lncRNA. Here, we present key themes within the field of lncRNAs, emphasising the importance of their roles in both the nucleus and the cytoplasm of cells, as well as patterns in their modes of action. We discuss their potential functions in development and disease using examples where we have the greatest understanding. Finally, we emphasise why lncRNAs can serve as biomarkers and discuss their emerging potential for therapy.No conflicts of interest were declared. What are lncRNAs?LncRNAs are RNAs of >200 nucleotides (nt) in length that are not thought to code for proteins. Although our appreciation and understanding of lncRNA function and importance has exploded in the last decade, the first lncRNAs were discovered in the 1990s: BC200, H19 [1], and Xist [2]. In the post-genomic era, extensive and deep RNA-Seq has revealed the existence of huge numbers of novel RNA transcripts, including lncRNAs. Many of these novel transcripts are low in abundance and so were not previously identified. Several consortia have been responsible for sequencing RNA from a variety of tissues, cell types, organisms, and disease states, and we now have a much more precise view of which RNA transcripts are expressed, and when and where (GENCODE [3], GTEX [4], FANTOM [5]).

Regulation of protein synthesis is a vital step in controlling gene expression, especially during development. Over the last 10 years, it has become clear that rather than being homogeneous machines responsible for mRNA translation, ribosomes are highly heterogeneous and can play an active part in translational regulation. These “specialized ribosomes” comprise of specific protein and/or rRNA components, which are required for the translation of particular mRNAs. However, while there is extensive evidence for ribosome heterogeneity, support for specialized functions is limited. Recent work in a variety of developmental model organisms has shed some light on the biological relevance of ribosome heterogeneity. Tissue‐specific expression of ribosomal components along with phenotypic analysis of ribosomal gene mutations indicate that ribosome heterogeneity and potentially specialization are common in key development processes like embryogenesis, spermatogenesis, oogenesis, body patterning, and neurogenesis. Several examples of ribosome specialization have now been proposed but strong links between ribosome heterogeneity, translation of specific mRNAs by defined mechanisms, and role of these translation events remain elusive. Furthermore, several studies have indicated that heterogeneous ribosome populations are a product of tissue‐specific expression rather than specialized function and that ribosomal protein phenotypes are the result of extra‐ribosomal function or overall reduced ribosome levels. Many important questions still need to be addressed in order to determine the functional importance of ribosome heterogeneity to development and disease, which is likely to vary across systems. It will be essential to dissect these issues to fully understand diseases caused by disruptions to ribosomal composition, such as ribosomopathies. This article is categorized under: Translation > Translation Regulation Translation > Ribosome Structure/Function RNA in Disease and Development > RNA in Development

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 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.