Mitochondrial dysfunction and calcium dysregulation in COQ8A-ataxia Purkinje neurons are rescued by CoQ10 treatment.

COQ8A-ataxia is a rare form of neurodegenerative disorder due to mutations in the COQ8A gene. The encoded mitochondrial protein is involved in the regulation of coenzyme Q10 biosynthesis. Previous studies on the constitutive Coq8a-/- mice indicated specific alterations of cerebellar Purkinje neurons involving altered electrophysiological function and dark cell degeneration. In the present manuscript, we extend our understanding of the contribution of Purkinje neuron dysfunction to the pathology. By generating a Purkinje-specific conditional COQ8A knockout, we demonstrate that loss of COQ8A in Purkinje neurons is the main cause of cerebellar ataxia. Furthermore, through in vivo and in vitro approaches, we show that COQ8A-depleted Purkinje neurons have abnormal dendritic arborizations, altered mitochondria function and intracellular calcium dysregulation. Furthermore, we demonstrate that oxidative phosphorylation, in particular Complex IV, is primarily altered at presymptomatic stages of the disease. Finally, the morphology of primary Purkinje neurons as well as the mitochondrial dysfunction and calcium dysregulation could be rescued by CoQ10 treatment, suggesting that CoQ10 could be a beneficial treatment for COQ8A-ataxia.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex.

The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.

The mitoXplorer 2.0 update: integrating and interpreting mitochondrial expression dynamics within a cellular context.

Mitochondria are subcellular organelles present in almost all eukaryotic cells, which play a central role in cellular metabolism. Different tissues, health and age conditions are characterized by a difference in mitochondrial structure and composition. The visual data mining platform mitoXplorer 1.0 was developed to explore the expression dynamics of genes associated with mitochondrial functions that could help explain these differences. It, however, lacked functions aimed at integrating mitochondria in the cellular context and thus identifying regulators that help mitochondria adapt to cellular needs. To fill this gap, we upgraded the mitoXplorer platform to version 2.0 (mitoXplorer 2.0). In this upgrade, we implemented two novel integrative functions, network analysis and transcription factor enrichment, to specifically help identify signalling or transcriptional regulators of mitochondrial processes. In addition, we implemented several other novel functions to allow the platform to go beyond simple data visualization, such as an enrichment function for mitochondrial processes, a function to explore time-series data, the possibility to compare datasets across species and an IDconverter to help facilitate data upload. We demonstrate the usefulness of these functions in three specific use cases. mitoXplorer 2.0 is freely available without login at http://mitoxplorer2.ibdm.univ-mrs.fr.

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance.

Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.

The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development.

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

mitoXplorer, a visual data mining platform to systematically analyze and visualize mitochondrial expression dynamics and mutations.

Mitochondria participate in metabolism and signaling. They adapt to the requirements of various cell types. Publicly available expression data permit to study expression dynamics of genes with mitochondrial function (mito-genes) in various cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for mito-genes. Here, we introduce the visual data mining platform mitoXplorer, which integrates expression and mutation data of mito-genes with a manually curated mitochondrial interactome containing ∼1200 genes grouped in 38 mitochondrial processes. User-friendly analysis and visualization tools allow to mine mitochondrial expression dynamics and mutations across various datasets from four model species including human. To test the predictive power of mitoXplorer, we quantify mito-gene expression dynamics in trisomy 21 cells, as mitochondrial defects are frequent in trisomy 21. We uncover remarkable differences in the regulation of the mitochondrial transcriptome and proteome in one of the trisomy 21 cell lines, caused by dysregulation of the mitochondrial ribosome and resulting in severe defects in oxidative phosphorylation. With the newly developed Fiji plugin mitoMorph, we identify mild changes in mitochondrial morphology in trisomy 21. Taken together, mitoXplorer (http://mitoxplorer.ibdm.univ-mrs.fr) is a user-friendly, web-based and freely accessible software, aiding experimental scientists to quantify mitochondrial expression dynamics.

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.

A cell cycle-regulated Slx4-Dpb11 complex promotes the resolution of DNA repair intermediates linked to stalled replication.

A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.

Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC.

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15-null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.

Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture.

Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. Here, we establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.

The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury.

BACKGROUND: Although AKI lacks effective therapeutic approaches, preventive strategies using preconditioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. METHODS: To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. RESULTS: The gene expression signatures induced by both preconditioning strategies involve distinct common genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. CONCLUSIONS: This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny.cecad.uni-koeln.de:3838/IRaP/).

DNA-protein crosslink repair: proteases as DNA repair enzymes.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions because they interfere with DNA transactions. The recent discovery of a yeast protease that processes DPCs proteolytically raises the question whether DPC proteases also exist in higher eukaryotes. We argue here that the yeast enzyme, Wss1 (weak suppressor of smt3), is a member of a protease family whose mammalian representative is Spartan (SprT-like domain-containing protein)/DVC1 (DNA damage protein targeting VCP). DPC proteases may thus be common to all eukaryotes where they function as novel guardians of the genome.

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis.

BACKGROUND & AIMS: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. Deregulation of receptor tyrosine kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. METHODS: A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose mice to tumorigenesis. Systematic sequencing of tumours identified common transposon insertion sites, thus uncovering putative RTK cooperators for liver cancer. Bioinformatic analyses were applied to transposon outcomes and human HCC datasets. In vitro and in vivo (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. RESULTS: We identified 275 genes, most of which are altered in patients with HCC. Unexpectedly, these genes are not restricted to a small set of pathway/cellular processes, but cover a large spectrum of cellular functions, including signalling, metabolism, chromatin remodelling, mRNA degradation, proteasome, ubiquitination, cell cycle regulation, and chromatid segregation. We validated 15 tumour suppressor candidates, as shRNA-mediated targeting confers tumorigenicity to RTK-sensitized cells, but not to cells with basal RTK levels. This demonstrates that the context of enhanced RTK levels is essential for their action in tumour initiation. CONCLUSION: Our study identifies unanticipated genetic interactions underlying gene cooperativity with RTKs in HCC. Moreover, these results show how subtly increased levels of wild-type RTKs provide a tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. LAY SUMMARY: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. We have applied a genetic method to randomly mutate large numbers of genes in the context of a mouse model with increased RTK levels, predisposed to develop liver cancer. We identified mechanisms that accelerate tumour formation in cooperation with enhanced RTK levels. The wide array of cellular functions among these cooperators illustrates an extraordinary capability of RTKs to render the liver more vulnerable to additional alterations, by priming cells for tumour initiation.

HH-MOTiF: de novo detection of short linear motifs in proteins by Hidden Markov Model comparisons.

Short linear motifs (SLiMs) in proteins are self-sufficient functional sequences that specify interaction sites for other molecules and thus mediate a multitude of functions. Computational, as well as experimental biological research would significantly benefit, if SLiMs in proteins could be correctly predicted de novo with high sensitivity. However, de novo SLiM prediction is a difficult computational task. When considering recall and precision, the performances of published methods indicate remaining challenges in SLiM discovery. We have developed HH-MOTiF, a web-based method for SLiM discovery in sets of mainly unrelated proteins. HH-MOTiF makes use of evolutionary information by creating Hidden Markov Models (HMMs) for each input sequence and its closely related orthologs. HMMs are compared against each other to retrieve short stretches of homology that represent potential SLiMs. These are transformed to hierarchical structures, which we refer to as motif trees, for further processing and evaluation. Our approach allows us to identify degenerate SLiMs, while still maintaining a reasonably high precision. When considering a balanced measure for recall and precision, HH-MOTiF performs better on test data compared to other SLiM discovery methods. HH-MOTiF is freely available as a web-server at http://hh-motif.biochem.mpg.de.

SLALOM, a flexible method for the identification and statistical analysis of overlapping continuous sequence elements in sequence- and time-series data.

BACKGROUND: Protein or nucleic acid sequences contain a multitude of associated annotations representing continuous sequence elements (CSEs). Comparing these CSEs is needed, whenever we want to match identical annotations or integrate distinctive ones. Currently, there is no ready-to-use software available that provides comprehensive statistical readout for comparing two annotations of the same type with each other, which can be adapted to the application logic of the scientific question. RESULTS: We have developed a method, SLALOM (for StatisticaL Analysis of Locus Overlap Method), to perform comparative analysis of sequence annotations in a highly flexible way. SLALOM implements six major operation modes and a number of additional options that can answer a variety of statistical questions about a pair of input annotations of a given sequence collection. We demonstrate the results of SLALOM on three different examples from biology and economics and compare our method to already existing software. We discuss the importance of carefully choosing the application logic to address specific scientific questions. CONCLUSION: SLALOM is a highly versatile, command-line based method for comparing annotations in a collection of sequences, with a statistical read-out for performance evaluation and benchmarking of predictors and gene annotation pipelines. Abstraction from sequence content even allows SLALOM to compare other kinds of positional data including, for example, data coming from time series.

Integrative analysis and machine learning on cancer genomics data using the Cancer Systems Biology Database (CancerSysDB).

BACKGROUND: Recent cancer genome studies on many human cancer types have relied on multiple molecular high-throughput technologies. Given the vast amount of data that has been generated, there are surprisingly few databases which facilitate access to these data and make them available for flexible analysis queries in the broad research community. If used in their entirety and provided at a high structural level, these data can be directed into constantly increasing databases which bear an enormous potential to serve as a basis for machine learning technologies with the goal to support research and healthcare with predictions of clinically relevant traits. RESULTS: We have developed the Cancer Systems Biology Database (CancerSysDB), a resource for highly flexible queries and analysis of cancer-related data across multiple data types and multiple studies. The CancerSysDB can be adopted by any center for the organization of their locally acquired data and its integration with publicly available data from multiple studies. A publicly available main instance of the CancerSysDB can be used to obtain highly flexible queries across multiple data types as shown by highly relevant use cases. In addition, we demonstrate how the CancerSysDB can be used for predictive cancer classification based on whole-exome data from 9091 patients in The Cancer Genome Atlas (TCGA) research network. CONCLUSIONS: Our database bears the potential to be used for large-scale integrative queries and predictive analytics of clinically relevant traits.

The deregulated microRNAome contributes to the cellular response to aneuploidy.

BACKGROUND: Aneuploidy, or abnormal chromosome numbers, severely alters cell physiology and is widespread in cancers and other pathologies. Using model cell lines engineered to carry one or more extra chromosomes, it has been demonstrated that aneuploidy per se impairs proliferation, leads to proteotoxic as well as replication stress and triggers conserved transcriptome and proteome changes. RESULTS: In this study, we analysed for the first time miRNAs and demonstrate that their expression is altered in response to chromosome gain. The miRNA deregulation is independent of the identity of the extra chromosome and specific to individual cell lines. By cross-omics analysis we demonstrate that although the deregulated miRNAs differ among individual aneuploid cell lines, their known targets are predominantly associated with cell development, growth and proliferation, pathways known to be inhibited in response to chromosome gain. Indeed, we show that up to 72% of these targets are downregulated and the associated miRNAs are overexpressed in aneuploid cells, suggesting that the miRNA changes contribute to the global transcription changes triggered by aneuploidy. We identified hsa-miR-10a-5p to be overexpressed in majority of aneuploid cells. Hsa-miR-10a-5p enhances translation of a subset of mRNAs that contain so called 5'TOP motif and we show that its upregulation in aneuploids provides resistance to starvation-induced shut down of ribosomal protein translation. CONCLUSIONS: Our work suggests that the changes of the microRNAome contribute on one hand to the adverse effects of aneuploidy on cell physiology, and on the other hand to the adaptation to aneuploidy by supporting translation under adverse conditions.