Age-dependent gain of alternative splice forms and biased duplication explain the relation between splicing and duplication.

We analyze here the relation between alternative splicing and gene duplication in light of recent genomic data, with a focus on the human genome. We show that the previously reported negative correlation between level of alternative splicing and family size no longer holds true. We clarify this pattern and show that it is sufficiently explained by two factors. First, genes progressively gain new splice variants with time. The gain is consistent with a selectively relaxed regime, until purifying selection slows it down as aging genes accumulate a large number of variants. Second, we show that duplication does not lead to a loss of splice forms, but rather that genes with low levels of alternative splicing tend to duplicate more frequently. This leads us to reconsider the role of alternative splicing in duplicate retention.

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

Splice variant-specific stabilization of JNKs by IB1/JIP1.

Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that tethers components of the JNK mitogen-activated protein kinase pathway inducing a modulation of the activity and the target specificity of the JNK kinases. Dysfunctions in IB1 have been associated with diseases such as early type II diabetes. To gain more insight in the functions of IB1, its ability to modulate the expression levels of the various JNK proteins was assessed. Each of the three JNK genes gives rise to several splice variants encoding short or long proteins. The expression levels of the short JNK proteins, but not of the long variants, were systematically higher in rat tissues and in transformed cell lines expressing high IB1 levels compared to tissues and cells with no or low IB1 expression. HEK293 cells bearing a tetracycline-inducible IB1 construct showed a specific increase of the short JNK endogenous splice variants in the presence of tetracycline. The augmented expression level of the short JNK splice variants induced by IB1 resulted from an increased stability towards degradation. Modulation of the stability of specific JNK splice variants represents therefore a newly identified mechanism used by IB1 to regulate the JNK MAPK pathway.

The evolution of alternative splicing patterns and regulatory mechanisms

Alternative splicing produces multiple isoforms from the same gene, thus increasing the number of transcripts of the species. Alternative splicing is a virtually ubiquitous mechanism in eukaryotes, for example more than 90% of protein-coding genes in human are alternatively spliced. Recent evolutionary studies showed that alternative splicing is a fast evolving and highly species- specific mechanism. The rapid evolution of alternative splicing was considered as a contribution to the phenotypic diversity between species. However, the function of many isoforms produced by alternative splicing remains unclear and they might be the result of noisy splicing. Thus, the functional relevance of alternative splicing and the evolutionary mechanisms of its rapid divergence among species are still poorly understood. During my thesis, I performed a large-scale analysis of the regulatory mechanisms that drive the rapid evolution of alternative splicing. To study the evolution of alternative splicing regulatory mechanisms, I used an extensive RNA-sequencing dataset comprising 12 tetrapod species (human, chimpanzee and bonobo, gorilla, orangutan, macaque, marmoset, mouse, opossum, platypus, chicken and frog) and 8 tissues (cerebellum, brain, heart, kidney, liver, testis, placenta and ovary). To identify the catalogue of alternative splicing eis-acting regulatory elements in the different tetrapod species, I used a previously defined computational approach. This approach is a statistical analysis of exons/introns and splice sites composition and relies on a principle of compensation between splice sites strength and the presence of additional regulators. With an evolutionary comparative analysis of the exonic eis-acting regulators, I showed that these regulatory elements are generally shared among primates and more conserved than non-regulatory elements. In addition, I showed that the usage of these regulatory elements is also more conserved than expected by chance. In addition to the identification of species- specific eis-acting regulators, these results may explain the rapid evolution of alternative splicing. I also developed a new approach based on evolutionary sequence changes and corresponding alternative splicing changes to identify potential splicing eis-acting regulators in primates. The identification of lineage-specific substitutions and corresponding lineage-specific alternative splicing changes, allowed me to annotate the genomic sequences that might have played a role in the alternative splicing pattern differences among primates. Finally, I showed that the identified splicing eis-acting regulator datasets are enriched in human disease-causing mutations, thus confirming their biological relevance.

Traitement de la schistosomiase à S. mansoni : quelle alternative au praziquantel ?

Les schistosomiases sont des maladies parasitaires causées par des helminthes du genre Schistosoma (S.) qui touchent 200 millions de personnes dans le monde, mais restent rares chez le voyageur. Contrairement à S. heamatobium, agent de la bilharziose urinaire, S. mansoni, présent en Afrique subsaharienne, en Egypte ainsi qu'aux Antilles, au Surinam et dans le nordest du Brésil, est responsable des formes hépato-intestinales de la maladie. Les larves, vivant en eaux douces contaminées par des selles infectées, peuvent pénétrer la peau des baigneurs sans que l'individu ne s'en rende compte. Les parasites adultes s'établissent dans le système veineux digestif où ils se reproduisent et excrètent des oeufs qui migreront dans la lumière intestinale. Cette revue systématique évalue les effets des médicaments antibilharziens, utilisés seuls ou en association, pour traiter l'infection à S. mansoni.

Mass spectrometry as a rapid and powerful alternative to antibodies for detecting LPXTG wall-associated proteins of Staphylococcus aureus

Functional characterization of transformed or natively present bacterial virulence proteins can be achieved employing various model systems. A prerequisite is to verify the correct expression of the transformed protein or the presence of the native protein in the microbe. Traditionally, antibodies are raised against the protein or a peptide thereof, followed by Western blot analysis or by fluorescence-activated cell sorting. Alternatively, the protein-coding gene can be fused with a downstream reporter gene, the expression of which reports the simultaneous expression of the upstream recombinant protein. Although being powerful, these methods are time consuming, especially when multiple proteins must be assessed. Here we describe a novel way to validate the expression of Gram-positive surface proteins covalently attached to the peptidoglycan. Eighteen out of the 21 known LPXTG-motif carrying cell wall-associated proteins of Staphylococcus aureus were cloned in Lactoccocus lactis either alone, in combinations or as truncated forms, and their correct expression was assessed by liquid chromatography coupled to mass spectrometry (LC-MS). The method is rapid, sensitive and precise. It can identify multiple proteins in transformed constructs without the time and cost needed for raising and testing multiple sets of antibodies.

Which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (TNF) agents after previous failure of an anti-TNF agent?

BACKGROUND: Patients with rheumatoid arthritis (RA) with an inadequate response to TNF antagonists (aTNFs) may switch to an alternative aTNF or start treatment from a different class of drugs, such as rituximab (RTX). It remains unclear in which clinical settings these therapeutic strategies offer most benefit. OBJECTIVE: To analyse the effectiveness of RTX versus alternative aTNFs on RA disease activity in different subgroups of patients. METHODS: A prospective cohort study of patients with RA who discontinued at least one aTNF and subsequently received either RTX or an alternative aTNF, nested within the Swiss RA registry (SCQM-RA) was carried out. The primary outcome, longitudinal improvement in 28-joint count Disease Activity Score (DAS28), was analysed using multivariate regression models for longitudinal data and adjusted for potential confounders. RESULTS: Of the 318 patients with RA included; 155 received RTX and 163 received an alternative aTNF. The relative benefit of RTX varied with the type of prior aTNF failure: when the motive for switching was ineffectiveness to previous aTNFs, the longitudinal improvement in DAS28 was significantly better with RTX than with an alternative aTNF (p = 0.03; at 6 months, -1.34 (95% CI -1.54 to -1.15) vs -0.93 (95% CI -1.28 to -0.59), respectively). When the motive for switching was other causes, the longitudinal improvement in DAS28 was similar for RTX and alternative aTNFs (p = 0.40). These results were not significantly modified by the number of previous aTNF failures, the type of aTNF switches, or the presence of co-treatment with a disease-modifying antirheumatic drug. CONCLUSION: This observational study suggests that in patients with RA who have stopped a previous aTNF treatment because of ineffectiveness changing to RTX is more effective than switching to an alternative aTNF.

Synergistic epistasis and alternative hypotheses.

Inbreeding generally results in deleterious shifts in mean fitness. If the fitness response to increasing inbreeding coefficient is non-linear, this suggests a contribution of epistasis to inbreeding depression. In a cross-breeding experiment, Salathe & Ebert (2003. J. Evol. Biol. 16: 976-985) tested and found the presence of this non-linearity in Daphnia magna. They argue that epistatic interactions cause this non-linearity. We argue here that their experimental protocol does not allow disentangling the effect of synergistic epistasis from two alternative hypotheses, namely hybrid vigour and statistical non-independence of data.

Disease-causing mutations improving the branch site and polypyrimidine tract: pseudoexon activation of LINE-2 and antisense Alu lacking the poly(T)-tail.

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes.

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12 degreesC). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.