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BACKGROUND: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. RESULTS: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. CONCLUSIONS: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent., We acknowledge the contributions of Maximilian Hecht, Alexander Grün, Julia Krumhoff, My Nguyen Ly, Jonathan Boidol, Rene Schoeffel, Yann Spöri, Jessika Binder, Christoph Hamm and Karolina Worf. This work was partially supported by the following grants: National Science Foundation grants DBI-1458477 (PR), DBI-1458443 (SDM), DBI-1458390 (CSG), DBI-1458359 (IF), IIS-1319551 (DK), DBI-1262189 (DK), and DBI-1149224 (JC); National Institutes of Health grants R01GM093123 (JC), R01GM097528 (DK), R01GM076990 (PP), R01GM071749 (SEB), R01LM009722 (SDM), and UL1TR000423 (SDM); the National Natural Science Foundation of China grants 3147124 (WT) and 91231116 (WT); the National Basic Research Program of China grant 2012CB316505 (WT); NSERC grant RGPIN 371348-11 (PP); FP7 infrastructure project TransPLANT Award 283496 (ADJvD); Microsoft Research/FAPESP grant 2009/53161-6 and FAPESP fellowship 2010/50491-1 (DCAeS); Biotechnology and Biological Sciences Research Council grants BB/L020505/1 (DTJ), BB/F020481/1 (MJES), BB/K004131/1 (AP), BB/F00964X/1 (AP), and BB/L018241/1 (CD); the Spanish Ministry of Economics and Competitiveness grant BIO2012-40205 (MT); KU Leuven CoE PFV/10/016 SymBioSys (YM); the Newton International Fellowship Scheme of the Royal Society grant NF080750 (TN). CSG was supported in part by the Gordon and Betty Moore Foundation’s Data-Driven Discovery Initiative grant GBMF4552. Computational resources were provided by CSC – IT Center for Science Ltd., Espoo, Finland (TS). This work was supported by the Academy of Finland (TS). RCL and ANM were supported by British Heart Foundation grant RG/13/5/30112. PD, RCL, and REF were supported by Parkinson’s UK grant G-1307, the Alexander von Humboldt Foundation through the German Federal Ministry for Education and Research, Ernst Ludwig Ehrlich Studienwerk, and the Ministry of Education, Science and Technological Development of the Republic of Serbia grant 173001. This work was a Technology Development effort for ENIGMA – Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, which is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research grant DE-AC02-05CH11231. ENIGMA only covers the application of this work to microbial proteins. NSF DBI-0965616 and Australian Research Council grant DP150101550 (KMV). NSF DBI-0965768 (ABH). NIH T15 LM00945102 (training grant for CSF). FP7 FET grant MAESTRA ICT-2013-612944 and FP7 REGPOT grant InnoMol (FS). NIH R01 GM60595 (PCB). University of Padova grants CPDA138081/13 (ST) and GRIC13AAI9 (EL). Swiss National Science Foundation grant 150654 and UK BBSRC grant BB/M015009/1 (COD). PRB2 IPT13/0001 - ISCIII-SGEFI / FEDER (JMF)., This is the final version of the article. It first appeared from BioMed Central at http://dx.doi.org/10.1186/s13059-016-1037-6.

This study explored why lesioned retinal ganglion cell (RGC) axons regenerate successfully in the zebrafish optic nerve despite the presence of Rtn4b, the homologue of the rat neurite growth inhibitor RTN4-A/Nogo-A. Rat Nogo-A and zebrafish Rtn4b possess characteristic motifs (M1-4) in the Nogo-A-specific region, which contains delta20, the most inhibitory region of rat Nogo-A. To determine whether zebrafish M1-4 is inhibitory as rat M1-4 and Nogo-A delta20, proteins were recombinantly expressed and used as substrates for zebrafish single cell RGCs, mouse hippocampal neurons and goldfish, zebrafish and chick retinal explants. When offered as homogenous substrates, neurites of hippocampal neurons and of zebrafish single cell RGCs were inhibited by zebrafish M1-4, rat M1-4, and Nogo-A delta20. Neurite length increased when zebrafish single cell RGCs were treated with receptor-type-specific antagonists and, respectively, with morpholinos (MO) against S1PR2 and S1PR5a-which represent candidate zebrafish Nogo-A receptors. In a stripe assay, however, where M1-4 lanes alternate with polylysine-(Plys)-only lanes, RGC axons from goldfish, zebrafish, and chick retinal explants avoided rat M1-4 but freely crossed zebrafish M1-4 lanes-suggesting that zebrafish M1-4 is growth permissive and less inhibitory than rat M1-4. Moreover, immunostainings and dot blots of optic nerve and myelin showed that expression of Rtn4b is very low in tissue and myelin at 3-5 days after lesion when axons regenerate. Thus, Rtn4b seems to represent no major obstacle for axon regeneration in vivo because it is less inhibitory for RGC axons from retina explants, and because of its low abundance. published

The Gene Ontology Annotation (GOA) resource (http://www.ebi.ac.uk/GOA) provides evidence-based Gene Ontology (GO) annotations to proteins in the UniProt Knowledgebase (UniProtKB). Manual annotations provided by UniProt curators are supplemented by manual and automatic annotations from model organism databases and specialist annotation groups. GOA currently supplies 368 million GO annotations to almost 54 million proteins in more than 480,000 taxonomic groups. The resource now provides annotations to five times the number of proteins it did 4 years ago. As a member of the GO Consortium, we adhere to the most up-to-date Consortium-agreed annotation guidelines via the use of quality control checks that ensures that the GOA resource supplies high-quality functional information to proteins from a wide range of species. Annotations from GOA are freely available and are accessible through a powerful web browser as well as a variety of annotation file formats.

The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads.

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5 mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40–60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.

Unlike mammals, fish are able to regenerate axons in their central nervous system. This difference has been partly attributed to the loss/acquisition of inhibitory proteins during evolution. Nogo-A--the longest isoform of the reticulon4 (rtn4) gene product--is commonly found in mammalian myelin where it acts as a potent inhibitor of axonal regeneration. Interestingly, fish RTN4 isoforms were previously reported to lack the most inhibitory Nogo-A-specific region (NSR). Nevertheless, fish axons collapse on contact with mammalian NSR, suggesting that fish possess a functional Nogo-A receptor but not its ligand. To reconcile these findings, we revisited the early evolution of rtn4. Mining of current genome databases established the unequivocal presence of NSR-coding sequences in fish rtn4 paralogues. Further comparative analyses indicate that the common ancestor of fish and tetrapods had an NSR-coding rtn4 gene, which underwent duplication and divergent evolution in bony fish. Our genomic survey also revealed that the cephalochordate Branchiostoma floridae contains a single rtn gene lacking the NSR. Hence, Nogo-A most probably arose independently in the rtn4 gene of a gnathostome ancestor before the split of the fish and tetrapod lineages. Close examination of the NSR uncovered clusters of structural and sequential similarities with neurocan (NCAN), an inhibitory proteoglycan of the glial scar. Notably, the shared presence of transposable elements in ncan and rtn4 genes suggests that Nogo-A originated via insertion of an ncan-like sequence into the rtn4 gene of an early jawed vertebrate with myelinated axons.

In contrast to fish, mammals are unable to regenerate lesioned fiber tracts in the central nervous system (CNS). Two major factors are postulated to be responsible: intrinsic neuronal properties and the glial cell environment. Upon injury several types of molecules produced by glial cells elicit inhibitory effect on neurite outgrowth. One of these, oligodendrocyte-derived RTN4/Nogo was demonstrated to act as a potent inhibitor of axon regeneration and to block neurite extension via two domains: the Nogo-A-specific region and Nogo-66 located in N- and C-terminal parts of the protein, respectively.The present work focuses on the evolution of RTN4/Nogo and its inhibitory domains in chordates as well as their functional characterization in fish. In order to comprehend the origin of the Nogo-A-specific region (NSR) all available genomes of chordates were analyzed. Our data indicate that the common ancestor of fish and tetrapods had an NSR-coding rtn4 gene, which underwent duplication and divergent evolution in bony fish. Thus, in the zebrafish, the NSR was lost in rtn4 but retained in its duplicate rtn6, whereas in the pufferfish, the NSR was retained in rtn4 and the entire rtn6 gene was deleted. Distant homology screening in combination with protein architecture analysis reveals the relation of this region to CSPG neurocan on the levels of domain organization and sequence similarity, such as shared presence of the putative integrin-binding motifs. Therefore, Nogo-A most likely originated from the insertion of a neurocan DNA sequence into an ancestral rtn4 gene. Notably, the proposed timing of this event coincides with the acquisition of jaws and myelin by vertebrates. These results not only shed light on the evolution of Nogo-A, but may facilitate the identification of its molecular receptor(s).Although the NSRs in fish and mammals share only 18% identity on the primary structure level, zebrafish Nogo-66 is 66% identical and more than 80% similar to its rat homologue. This notion raises the question whether the fish peptide is able to exert neurite outgrowth inhibition. Surprisingly, in the outgrowth, collapse and contact assays zebrafish Nogo-66 appeared to be growth-permissive for fish and mammalian neurons, quite in contrast to its rat Nogo-66 homologue which inhibits growth. Upon binding to their common receptor NgR1, the rat peptide in contrast to zebrafish Nogo-66 elicits phosphorylation of the downstream effector cofilin which leads to actin filament disassembly. These data are in agreement with the apparent absence of neurite outgrowth inhibitors in fish CNS. However, it is not clear how so similar peptides can exert different responses. Thus, we have analyzed Nogo-66/NgR1 interaction combining coevolutionary and structure modeling approaches. Our results demonstrate that both proteins are already present in cephalochordates but began to coevolve only after fish-tetrapod split. Based on conservation analysis of primary and tertiary structures of these molecules and on published functional data we were able to reconstruct the receptor-ligand complex and model Nogo-66/NgR1 interactions in mammals and fish. The obtained results are in agreement with the previous notion that Nogo-66/NgR1-induced signal transduction becomes inhibitory during/after the fish-tetrapod transition.These extensive analyses of evolution of both inhibitory domains of RTN4/Nogo may help to understand why the ability to regenerate lesioned axons in the CNS became restricted during vertebrate evolution. Moreover, the combination of structural and functional approaches can provide the necessary information which molecular changes during RTN4 evolution are responsible for the acquisition of its inhibitory properties.

In contrast to mammals, lesioned axons in the zebrafish (ZF) optic nerve regenerate and restore vision. This correlates with the absence of the NogoA-specific N-terminal domains from the ZFnogo/rtn-4(reticulon-4) gene that inhibits regeneration in mammals. However, mammaliannogo/rtn-4carries a second inhibitory C-terminal domain, Nogo-66, being 70% identical with ZF-Nogo66. The present study examines, (1) whether ZF-Nogo66 is inhibitory and effecting similar signaling pathways upon Nogo66-binding to the Nogo66 receptor NgR and its coreceptors, and (2) whether Rat-Nogo66 on fish, and ZF-Nogo66 on mouse neurons, cause inhibition via NgR. Our results from “outgrowth, collapse and contact assays” suggest, surprisingly, that ZF-Nogo66 is growth-permissive for ZF and mouse neurons, quite in contrast to its Rat-Nogo66 homolog which inhibits growth. The opposite effects of ZF- and Rat-Nogo66 are, in both fish and mouse, transmitted by GPI (glycosylphosphatidylinositol)-anchored receptors, including NgR. The high degree of sequence homology in the predicted binding site is consistent with the ability of ZF- and mammalian-Nogo66 to bind to NgRs of both species. Yet, Rat-Nogo66 elicits phosphorylation of the downstream effector cofilin whereas ZF-Nogo66 has no influence on cofilin phosphorylation—probably because of significantly different Rat- versus ZF-Nogo66 sequences outside of the receptor-binding region effecting, by speculation, recruitment of a different set of coreceptors or microdomain association of NgR. Thus, not only was the NogoA-specific domain lost in fish, but Nogo66, the second inhibitory domain in mammals, and its signaling upon binding to NgR, was modified so that ZF-Nogo/RTN-4 does not impair axon regeneration.