Non-EST based prediction of exon skipping and intron retention events using Pfam information

Most of the known alternative splice events have been detected by the comparison of expressed sequence tags (ESTs) and cDNAs. However, not all splice events are represented in EST databases since ESTs have several biases. Therefore, non-EST based approaches are needed to extend our view of a transcriptome. Here, we describe a novel method for the ab initio prediction of alternative splice events that is solely based on the annotation of Pfam domains. Furthermore, we applied this approach in a genome-wide manner to all human RefSeq transcripts and predicted a total of 321 exon skipping and intron retention events. We show that this method is very reliable as 78% (250 of 321) of our predictions are confirmed by ESTs or cDNAs. Subsequent analyses of splice events within Pfam domains revealed a significant preference of alternative exon junctions to be located at the protein surface and to avoid secondary structure elements. Thus, splice events within Pfams are probable to alter the structure and function of a domain which makes them highly interesting for detailed biological investigation. As Pfam domains are annotated in many other species, our strategy to predict exon skipping and intron retention events might be important for species with a lower number of ESTs

Posttranslational Modification of Human Glyoxalase 1 Indicates Redox-Dependent Regulation

Background: Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic a-oxo-aldehydes into the corresponding a-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown. Methodology/Principal Findings: We characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys 19/20, did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation. Conclusion/Significance: It is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within th

TassDB: a database of alternative tandem splice sites

Subtle alternative splice events at tandem splice sites are frequent in eukaryotes and substantially increase the complexity of transcriptomes and proteomes. We have developed a relational database, TassDB (TAndem Splice Site DataBase), which stores extensive data about alternative splice events at GYNGYN donors and NAGNAG acceptors. These splice events are of subtle nature since they mostly result in the insertion/deletion of a single amino acid or the substitution of one amino acid by two others. Currently, TassDB contains 114 554 tandem splice sites of eight species, 5209 of which have EST/mRNA evidence for alternative splicing. In addition, human SNPs that affect NAGNAG acceptors are annotated. The database provides a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download large datasets. This database should facilitate further experimental studies and large-scale bioinformatics analyses of tandem splice sites. The database is available at

The Mating Pattern of Captive Naked Mole-Rats Is Best Described by a Monogamy Model

Naked mole-rats form colonies with a single reproductively active female surrounded by subordinate workers. Workers perform offspring care, construction and defense of the burrow system, and food supply. Such division of labor, called “cooperative breeding,” is strongly associated with the evolution of monogamous mating behavior, as seen in several mammalian lineages. This association is explained by the evolutionary theory of kin selection, according to which a subordinate adult may help to raise other’s offspring if they are in full sibling relationship. In conflict with this theory, the naked mole-rat is widely considered to be polyandrous, based on reports on multiple males contributing to a colony’s progeny. In order to resolve this contrast, we undertook an in-depth microsatellite-based kinship analysis on captive colonies. Four independent colonies comprising a total of 265 animals were genotyped using a panel of 73 newly established microsatellite markers. Our results show that each mole-rat colony contains a single monogamous breeder pair, which translates to a reproductive skew of 100% for both sexes. This finding, also in conjunction with previously published parental data, favors monogamy as the best-fitting model to describe naked mole-rat reproduction patterns. Polyandry or other polygamous reproduction models are disfavored and should be considered as exceptional. Overall, the empirical genetic data are in agreement with the kin selection theory.Peer Reviewe

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

TG dinucleotides functioning as alternative 3' splice sites were identified and experimentally verified in 36 human genes

Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing

<p>Abstract</p> <p>Background</p> <p>The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.</p> <p>Results</p> <p>Six PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.</p> <p>Conclusion</p> <p>Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.</p

Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression

<p>Abstract</p> <p>Background</p> <p>NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood.</p> <p>Results</p> <p>We have identified two novel exons of the <it>NOD2 </it>gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa <it>in vivo</it>. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy.</p> <p>Conclusion</p> <p>The differential usage of two alternative promoters in the <it>NOD2 </it>gene leads to tissue-specific and context-dependent <it>NOD2 </it>transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.</p

Ethyl pyruvate

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and antiinflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well

TassDB2 - A comprehensive database of subtle alternative splicing events

Background: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus, a database which covers all confirmed cases of subtle alternative splicing as well as the numerous putative tandem splice sites (which might be confirmed once more transcript data becomes available), and allows to search for tandem splice sites with specific features and download the results, is a valuable resource for targeted experimental studies and large-scale bioinformatics analyses of tandem splice sites. Towards this goal we recently set up TassDB (Tandem Splice Site DataBase, version 1), which stores data about alternative splicing events at tandem splice sites separated by 3 nt in eight species. \ud Description: We have substantially revised and extended TassDB. The currently available version 2 contains extensive information about tandem splice sites separated by 2-12 nt for the human and mouse transcriptomes including data on the conservation of the tandem motifs in five vertebrates. TassDB2 offers a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download result datasets. For example, users can search for cases of alternative splicing where the proportion of EST/mRNA evidence supporting the minor isoform exceeds a specific threshold, or where the difference in splice site scores is specified by the user. The predicted impact of each event on the protein is also reported, along with information about being a putative target for the nonsense-mediated decay \ud (NMD) pathway. Links are provided to the UCSC genome browser and other external resources.\ud Conclusion: TassDB2, available via http://www.tassdb.info, provides comprehensive resources for researchers interested in both targeted experimental studies and large-scale bioinformatics analyses of short distance tandem splice sites.\ud \ud doi: 10.1186/1471-2105-11-216\u