Jose Manuel Rodriguez

GENCODE: The reference human genome annotation for The ENCODE Project

The GENCODE Consortium aims to identify all gene features in the human genome using a combination of computational analysis, manual annotation, and experimental validation. Since the first public release of this annotation data set, few new protein-coding loci have been added, yet the number of alternative splicing transcripts annotated has steadily increased. The GENCODE 7 release contains 20,687 protein-coding and 9640 long noncoding RNA loci and has 33,977 coding transcripts not represented in UCSC genes and RefSeq. It also has the most comprehensive annotation of long noncoding RNA (lncRNA) loci publicly available with the predominant transcript form consisting of two exons. We have examined the completeness of the transcript annotation and found that 35% of transcriptional start sites are supported by CAGE clusters and 62% of protein-coding genes have annotated polyA sites. Over one-third of GENCODE protein-coding genes are supported by peptide hits derived from mass spectrometry spectra submitted to Peptide Atlas. New models derived from the Illumina Body Map 2.0 RNA-seq data identify 3689 new loci not currently in GENCODE, of which 3127 consist of two exon models indicating that they are possibly unannotated long noncoding loci. GENCODE 7 is publicly available from gencodegenes.org and via the Ensembl and UCSC Genome Browsers.

Multiple evidence strands suggest that there may be as few as 19 000 human protein-coding genes

Determining the full complement of protein-coding genes is a key goal of genome annotation. The most powerful approach for confirming protein-coding potential is the detection of cellular protein expression through peptide mass spectrometry (MS) experiments. Here, we mapped peptides detected in seven large-scale proteomics studies to almost 60% of the protein-coding genes in the GENCODE annotation of the human genome. We found a strong relationship between detection in proteomics experiments and both gene family age and cross-species conservation. Most of the genes for which we detected peptides were highly conserved. We found peptides for >96% of genes that evolved before bilateria. At the opposite end of the scale, we identified almost no peptides for genes that have appeared since primates, for genes that did not have any protein-like features or for genes with poor cross-species conservation. These results motivated us to describe a set of 2001 potential non-coding genes based on features such as weak conservation, a lack of protein features, or ambiguous annotations from major databases, all of which correlated with low peptide detection across the seven experiments. We identified peptides for just 3% of these genes. We show that many of these genes behave more like non-coding genes than protein-coding genes and suggest that most are unlikely to code for proteins under normal circumstances. We believe that their inclusion in the human protein-coding gene catalogue should be revised as part of the ongoing human genome annotation effort.

JAM2 interacts with alpha4beta1. Facilitation by JAM3.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn2+-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and α4β1 in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an α4-specific inhibitor, to attenuate the Mn2+-enhanced component. Intriguingly, the engagement of α4β1 by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/α4β1 interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and α4β1counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on α4β1 interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

APPRIS: annotation of principal and alternative splice isoforms

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

Comparative Proteomics Reveals a Significant Bias Toward Alternative Protein Isoforms with Conserved Structure and Function

Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of “novel” and “putative” protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and strongly suggests that the translation of alternative transcripts may be subject to selective constraints.

Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC.

The 72-kDa zinc finger transcription factor PacC, distantly related to Ci/Gli developmental regulators, undergoes two-step proteolytic processing in response to alkaline ambient pH. “Signaling protease” cleavage of PacC72 removes a processing-inhibitory C-terminal domain, making its truncated PacC53 product accessible to a second “processing” protease, yielding PacC27. Features of the processing proteolysis suggested the proteasome as a candidate protease. We constructed, using gene replacements, two missense active site mutations in preB, the Aspergillus nidulans orthologue of Saccharomyces cerevisiae PRE2 encoding the proteasome β5 subunit. preB1K101A is lethal. Viable preB2K101R impairs growth and, like its equivalent pre2K108R in yeast, impairs chymotryptic activity. pre2K108R and preB2K101R active site mutations consistently shift position of the scissile bonds when PacC is processed in S. cerevisiae and A. nidulans, respectively, indicating that PacC must be a direct substrate of the proteasome. preB2K101R leads to a 2–3-fold elevation in NimE mitotic cyclin levels but appears to result in PacC instability, suggesting an altered balance between processing and degradation. preB2K101R compensates the marked impairment in PacC27 formation resulting from deletion of the processing efficiency determinant in PacC, further indicating direct proteasomal involvement in the formation of PacC27. Deletion of a Gly-Pro-Ala-rich region within this processing efficiency determinant markedly destabilizes PacC. Arg substitutions of Lys residues within this efficiency determinant and nearby show that they cooperate to promote PacC processing. A quadruple Lys-to-Arg substitution (4K→R) impairs formation of PacC27 and leads to persistence of PacC53. Wild-type PacC53 becomes multiply phosphorylated upon alkaline pH exposure. Processing-impaired 4K→R PacC53 becomes excessively phosphorylated.

The EMBRACE web service collection

The EMBRACE (European Model for Bioinformatics Research and Community Education) web service collection is the culmination of a 5-year project that set out to investigate issues involved in developing and deploying web services for use in the life sciences. The project concluded that in order for web services to achieve widespread adoption, standards must be defined for the choice of web service technology, for semantically annotating both service function and the data exchanged, and a mechanism for discovering services must be provided. Building on this, the project developed: EDAM, an ontology for describing life science web services; BioXSD, a schema for exchanging data between services; and a centralized registry (http://www.embraceregistry.net) that collects together around 1000 services developed by the consortium partners. This article presents the current status of the collection and its associated recommendations and standards definitions.

Most highly expressed protein-coding genes have a single dominant isoform.

Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level. We interrogate peptides from eight large-scale human proteomics experiments and databases and find that there is a single dominant protein isoform, irrespective of tissue or cell type, for the vast majority of the protein-coding genes in these experiments, in partial agreement with the conclusions from the most recent large-scale RNAseq study. Remarkably, the dominant isoforms from the experimental proteomics analyses coincided overwhelmingly with the reference isoforms selected by two completely orthogonal sources, the consensus coding sequence variants, which are agreed upon by separate manual genome curation teams, and the principal isoforms from the APPRIS database, predicted automatically from the conservation of protein sequence, structure, and function.

Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

CARGO: a web portal to integrate customized biological information

There is a huge quantity of information generated in Life Sciences, and it is dispersed in many databases and repositories. Despite the broad availability of the information, there is a great demand for methods that are able to look for, gather and display distributed data in a standardized and friendly way. CARGO (Cancer And Related Genes Online) is a configurable biological web portal designed as a tool to facilitate, integrate and visualize results from Internet resources, independently of their native format or access method. Through the use of small agents, called widgets, supported by a Rich Internet Application (RIA) paradigm based on AJAX, CARGO provides pieces of minimal, relevant and descriptive biological information. The tool is designed to be used by experimental biologists with no training in bioinformatics. In the current state, the system presents a list of human cancer genes. Available at http://cargo.bioinfo.cnio.es