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 first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.This work was supported by the following grants: NHGRIU54HG003273 to R.A.G; EU Marie Curie ITN #215781 “Evonet” to M.A.; a Wellcome Trust Value in People (VIP) award to C.B. and Wellcome Trust graduate studentship WT089615MA to J.E.G; Marine rhythms of Life” of the University of Vienna, an FWF (http://www.fwf.ac.at/) START award (#AY0041321) and HFSP (http://www.hfsp.org/) research grant (#RGY0082/2010) to KT-­‐R; MFPL Vienna International PostDoctoral Program for Molecular Life Sciences (funded by Austrian Ministry of Science and Research and City of Vienna, Cultural Department -­‐Science and Research to T.K; Direct Grant (4053034) of the Chinese University of Hong Kong to J.H.L.H.; NHGRI HG004164 to G.M.; Danish Research Agency (FNU), Carlsberg Foundation, and Lundbeck Foundation to C.J.P.G.; U.S. National Institutes of Health R01AI55624 to J.H.W.; Royal Society University Research fellowship to F.M.J.; P.D.E. was supported by the BBSRC via the Babraham Institute;This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pbio.100200

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

Impact of uORFs in mediating regulation of translation in stress conditions

Background: A large fraction of genes contains upstream ORFs (uORFs) in the 5' untranslated region (5'UTR). The translation of uORFs can inhibit the translation of the main coding sequence, for example by causing premature dissociation of the two ribosomal units or ribosome stalling. However, it is currently unknown if most uORFs are inhibitory or if this activity is restricted to specific cases. Here we interrogate ribosome profiling data from three different stress experiments in yeast to gain novel insights into this question. Results: By comparing ribosome occupancies in different conditions and experiments we obtain strong evidence that, in comparison to primary coding sequences (CDS), which undergo translational arrest during stress, the translation of uORFs is mostly unaffected by changes in the environment. As a result, the relative abundance of uORF-encoded peptides increases during stress. In general, the changes in the translational efficiency of regions containing uORFs do not seem to affect downstream translation. The exception are uORFs found in a subset of genes that are significantly up-regulated at the level of translation during stress; these uORFs tend to be translated at lower levels in stress conditions than in optimal growth conditions, facilitating the translation of the CDS during stress. We find new examples of uORF-mediated regulation of translation, including the Gcn4 functional homologue fil1 and ubi4 genes in S. pombe. Conclusion: We find evidence that the relative amount of uORF-encoded peptides increases during stress. The increased translation of uORFs is however uncoupled from the general CDS translational repression observed during stress. In a subset of genes that encode proteins that need to be rapidly synthesized upon stress uORFs act as translational switches.1. Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) grants BFU2015–65235-P and PGC2018–094091-B-I00, co-funded by Fondo Europeo de Desarrollo Regional (FEDER); 2. Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya, grant 2017SGR01020; 3. Spanish National Bioinformatics Institute (INB), funded by ISCIII and FEDER, grant PT17/0009/0014 of the PE I + D + i 2013–2016

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history

Presence and absence of immunity genes in different arthropods.

<p>Counts of immune genes are shown for <i>S. maritima</i>, <i>D. pulex</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-McTaggart1" target="_blank">[131]</a>, <i>A. mellifera</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-Evans1" target="_blank">[86]</a>, <i>T. castaneum</i>, <i>Anopheles gambiae</i>, and <i>D. melanogaster</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-Dasmahapatra1" target="_blank">[132]</a>. ∼, identity of the gene is uncertain; -, not investigated.</p

Conserved macro synteny signal between <i>S. maritima</i> and the chordate lancelet <i>B. floridae</i> clustered into ancestral linkage groups.

<p>Each dot represents a pair of genes, one in <i>B. floridae</i>, one in <i>S. maritima</i>, assigned to the same gene family by our orthology analysis. The ancestral linkage group identifiers refer to groups of scaffolds from the <i>S. maritima</i> (SmALG) or <i>B. floridae</i> (BfALG) assemblies, as detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s066" target="_blank">File S2</a>. The identification of ALGs is described in the SI. Note that two <i>S. maritima</i> scaffolds were divided across ALGs, and so appear multiple times in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s066" target="_blank">File S2</a>.</p

Arthropod phylogenetic tree (with nematode outgroup) showing selected events of gene loss, gene gain, and gene family expansions.

<p>Main taxa are listed on the tips, with representative species for which there is a fully sequenced genome listed below. Major nodes are also named. Data from the genome of <i>S. maritima</i> allow us to map when in arthropod evolution these events occurred, even when these events did not occur on the centipede lineage. A plausible node for the occurrence of each event is marked and colour-coded, with the possible range marked with a thin line of the same colour. The events, listed from left to right are: (1) Dscam alternative splicing as a strategy for increasing immune diversity is known from <i>D. melanogaster</i>, as well as the crustacean <i>D. pulex</i>, and thus probably evolved in the lineage leading to pancrustacea, after the split from centipedes. (2) Several wnt genes have been lost in holometabolous insects, leaving only seven of the 13 ancestral families. This loss occurred gradually over arthropod evolution, but reached its peak at the base of the Holometabola. (3) Selenoproteins are rare in insects. The presence of a large number of selenoproteins in <i>S. maritima</i> as well as in other non-insect arthropods suggests that the loss of many selenoproteins occurred at the base of the Insecta. (4) Expansion of chemosensory gene families occurred independently in different arthropod lineages as they underwent terrestrialisation. The OR family is expanded in insects only. (5) Chemosensory genes of the GR and IR genes have undergone a lineage specific expansion in the genome of <i>S. maritima</i>. As these are probably also linked with terrestrialisation we suggest that this expansion happened at the base of the Chilopoda, but it could have also occurred later in the lineage leading to <i>S. maritima</i>. (6) Cuticular proteins of the RR-1 family are numerous in the <i>S. maritima</i> genome. They are found in other arthropods, but not in chelicerates nor in any non-arthropod species. This suggests that the RR-1 subfamily evolved at the base of the Mandibulata. (7) The genome of <i>S. maritima</i> has a large complement of wnt genes, but is missing <i>wnt8</i>. Since this gene is found in the Diplopod <i>G. marginata</i> (a species without a fully sequenced genome), the loss most likely occurred at the base of the Chilopoda. (8) Unlike the situation in <i>D. melanogaster</i>, immune diversity in the <i>S. maritima</i> genome is achieved through multiple copies of the Dscam gene. This expansion of the family could have happened at any time after the split between Myriapoda and Pancrustacea. (9) Both circadian rhythm genes and many light receptors are missing in <i>S. maritima</i>. These losses are most likely due to the subterranean life style of geophilomorph centipedes and are probably specific to this group. However, we cannot rule out the possibility that they were lost somewhere in the lineage leading to myriapods. (10) The existence of JH signalling in <i>S. maritima</i> as well as in all other arthropods studied to date strengthens the idea that this signalling system evolved with the exoskeleton of arthropods, though its origins could be even more ancient and date back to the origin of moulting at the base of the Ecdysozoa.</p