European Molecular Biology Laboratory EMBL Heidelberg

Heidelberg, Germany

European Molecular Biology Laboratory EMBL Heidelberg

Heidelberg, Germany

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Hauer C.,University of Heidelberg | Hauer C.,Molecular Medicine Partnership Unit MMPU | Hauer C.,European Molecular Biology Laboratory EMBL Heidelberg | Sieber J.,University of Heidelberg | And 12 more authors.
Cell Reports | Year: 2016

The exon junction complex (EJC) connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3) emerges as the EJC subunit that is almost exclusively bound to sites 20-24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins. Exon junction complexes govern multiple critical decisions in posttranscriptional gene regulation. Using all four RNA binding subunits of the complex, Hauer et al. provide a comprehensive map of bona fide EJCs across a mammalian transcriptome and show enrichment on alternatively spliced mRNAs and underrepresentation on RNAs encoding ribosomal proteins. © 2016 The Author(s).

PubMed | European Molecular Biology Laboratory EMBL Heidelberg, University of Ljubljana, University of Heidelberg and 1 Molecular Medicine Partnership Unit MMPU
Type: | Journal: Nature communications | Year: 2015

Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.

Chen W.-H.,European Molecular Biology Laboratory EMBL Heidelberg | Van Noort V.,European Molecular Biology Laboratory EMBL Heidelberg | Lluch-Senar M.,Barcelona Institute of Science and Technology | Lluch-Senar M.,University Pompeu Fabra | And 17 more authors.
Nucleic Acids Research | Year: 2015

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '- omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GCcontent and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. © The Author(s) 2016.

Mazaheri F.,European Molecular Biology Laboratory EMBL Heidelberg | Breus O.,European Molecular Biology Laboratory EMBL Heidelberg | Durdu S.,Cell Biology and Biophysics Unit | Haas P.,Center for Organismal Studies | And 4 more authors.
Nature Communications | Year: 2014

The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons. © 2014 Macmillan Publishers Limited. All rights reserved.

Kluth M.,University of Hamburg | Hesse J.,University of Hamburg | Heinl A.,University of Hamburg | Krohn A.,University of Hamburg | And 14 more authors.
Modern Pathology | Year: 2013

6q12-22 is the second most commonly deleted genomic region in prostate cancer. Mapping studies have described a minimally deleted area at 6q15, containing MAP3K7/TAK1, which was recently shown to have tumor suppressive properties. To determine prevalence and clinical significance of MAP3K7 alterations in prostate cancer, a tissue microarray containing 4699 prostate cancer samples was analyzed by fluorescence in situ hybridization. Heterozygous MAP3K7 deletions were found in 18.48% of 2289 interpretable prostate cancers. MAP3K7 deletions were significantly associated with advanced tumor stage (P<0.0001), high Gleason grade (P<0.0001), lymph node metastasis (P<0.0108) and early biochemical recurrence (P<0.0001). MAP3K7 alterations were typically limited to the loss of one allele as homozygous deletions were virtually absent and sequencing analyses revealed no evidence for MAP3K7 mutations in 15 deleted and in 14 non-deleted cancers. There was a striking inverse association of MAP3K7 deletions and TMPRSS2:ERG fusion status with 26.7% 6q deletions in 1125 ERG-negative and 11.1% 6q deletions in 1198 ERG-positive cancers (P<0.0001). However, the strong prognostic role of 6q deletions was retained in both ERG-positive and ERG-negative cancers (P<0.0001 each). In summary, our study identifies MAP3K7 deletion as a prominent feature in ERG-negative prostate cancer with strong association to tumor aggressiveness. MAP3K7 alterations are typically limited to one allele of the gene. Together with the demonstrated tumor suppressive function in cell line experiments and lacking evidence for inactivation through hypermethylation, these results indicate MAP3K7 as a gene for which haploinsufficency is substantially tumorigenic. © 2013 USCAP, Inc.

Hovelmann F.,Humboldt University of Berlin | Hovelmann F.,European Molecular Biology Laboratory EMBL Heidelberg | Gaspar I.,European Molecular Biology Laboratory EMBL Heidelberg | Chamiolo J.,Humboldt University of Berlin | And 4 more authors.
Chemical Science | Year: 2015

The simultaneous imaging of different RNA molecules in homogeneous solution is a challenge and requires optimisation to enable unambiguous staining of intracellular RNA targets. Our approach relies on single dye forced intercalation (FIT) probes, in which a visco-sensitive reporter of the thiazole orange (TO) family serves as a surrogate nucleobase and provides enhancements of fluorescence upon hybridisation. Previous FIT probes spanned the cyan and green emission range. Herein, we report for the first time chromophores for FIT probes that emit in the red range (above 600 nm). Such probes are valuable to overcome cellular auto-fluorescent background and enable multiplexed detection. In order to find suitable chromophores, we developed a submonomer approach that facilitated the rapid analysis of different TO family dyes in varied sequence positions. A carboxymethylated 4,4′-methine linked cyanine, which we named quinoline blue (QB), provided exceptional response characteristics at the 605 nm emission maximum. Exceeding previously reported base surrogates, the emission of the QB nucleotide intensified by up to 195-fold upon binding of complementary RNA. Owing to large extinction coefficients and quantum yields (up to ε = 129.000 L mol-1 cm-1 and Φ = 0.47, respectively) QB-FIT probes enable imaging of intracellular mRNA. A mixture of BO-, TO- and QB-containing FIT probes allowed the simultaneous detection of three different RNA targets in homogenous solution. TO- and QB-FIT probes were used to localize oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by means of wash-free fluorescent in situ hybridisation and super resolution microscopy (STED). © The Royal Society of Chemistry 2016.

Chen W.-H.,European Molecular Biology Laboratory EMBL Heidelberg | Zhao X.-M.,Tongji University | van Noort V.,European Molecular Biology Laboratory EMBL Heidelberg | Bork P.,European Molecular Biology Laboratory EMBL Heidelberg
PLoS Computational Biology | Year: 2013

Mendelian disorders are often caused by mutations in genes that are not lethal but induce functional distortions leading to diseases. Here we study the extent of gene duplicates that might compensate genes causing monogenic diseases. We provide evidence for pervasive functional redundancy of human monogenic disease genes (MDs) by duplicates by manifesting 1) genes involved in human genetic disorders are enriched in duplicates and 2) duplicated disease genes tend to have higher functional similarities with their closest paralogs in contrast to duplicated non-disease genes of similar age. We propose that functional compensation by duplication of genes masks the phenotypic effects of deleterious mutations and reduces the probability of purging the defective genes from the human population; this functional compensation could be further enhanced by higher purification selection between disease genes and their duplicates as well as their orthologous counterpart compared to non-disease genes. However, due to the intrinsic expression stochasticity among individuals, the deleterious mutations could still be present as genetic diseases in some subpopulations where the duplicate copies are expressed at low abundances. Consequently the defective genes are linked to genetic disorders while they continue propagating within the population. Our results provide insight into the molecular basis underlying the spreading of duplicated disease genes. © 2013 Chen et al.

Vogt A.,Austrian Academy of Sciences | Vogt A.,European Molecular Biology Laboratory EMBL Heidelberg | Mochizuki K.,Austrian Academy of Sciences
PLoS Genetics | Year: 2013

The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p. © 2013 Vogt, Mochizuki.

Reversi A.,European Molecular Biology Laboratory EMBL Heidelberg | Loeser E.,European Molecular Biology Laboratory EMBL Heidelberg | Subramanian D.,European Molecular Biology Laboratory EMBL Heidelberg | Schultz C.,European Molecular Biology Laboratory EMBL Heidelberg | De Renzis S.,European Molecular Biology Laboratory EMBL Heidelberg
Journal of Cell Biology | Year: 2014

Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. Here, we show that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. We found that an increase in PI(4,5)P2 levels caused premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/P1(3,4,5)P2 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape. © 2014 Alcaraz et al.

Fabrowski P.,European Molecular Biology Laboratory EMBL Heidelberg | Necakov A.S.,European Molecular Biology Laboratory EMBL Heidelberg | Mumbauer S.,European Molecular Biology Laboratory EMBL Heidelberg | Loeser E.,European Molecular Biology Laboratory EMBL Heidelberg | And 4 more authors.
Nature Communications | Year: 2013

During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development. © 2009-2012 IEEE.

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