The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.Oncogene advance online publication, 25 July 2016; doi:10.1038/onc.2016.260

Sequence information gain based motif analysis

Background: The detection of regulatory regions in candidate sequences is essential for the understanding of the regulation of a particular gene and the mechanisms involved. This paper proposes a novel methodology based on information theoretic metrics for finding regulatory sequences in promoter regions. Results: This methodology (SIGMA) has been tested on genomic sequence data for Homo sapiens and Mus musculus. SIGMA has been compared with different publicly available alternatives for motif detection, such as MEME/MAST, Biostrings (Bioconductor package), MotifRegressor, and previous work such Qresiduals projections or information theoretic based detectors. Comparative results, in the form of Receiver Operating Characteristic curves, show how, in 70 % of the studied Transcription Factor Binding Sites, the SIGMA detector has a better performance and behaves more robustly than the methods compared, while having a similar computational time. The performance of SIGMA can be explained by its parametric simplicity in the modelling of the non-linear co-variability in the binding motif positions. Conclusions: Sequence Information Gain based Motif Analysis is a generalisation of a non-linear model of the cis-regulatory sequences detection based on Information Theory. This generalisation allows us to detect transcription factor binding sites with maximum performance disregarding the covariability observed in the positions of the training set of sequences. SIGMA is freely available to the public at http://b2slab.upc.edu.Postprint (published version

PPIcons: identification of protein-protein interaction sites in selected organisms

The physico-chemical properties of interaction interfaces have a crucial role in characterization of protein–protein interactions (PPI). In silico prediction of participating amino acids helps to identify interface residues for further experimental verification using mutational analysis, or inhibition studies by screening library of ligands against given protein. Given the unbound structure of a protein and the fact that it forms a complex with another known protein, the objective of this work is to identify the residues that are involved in the interaction. We attempt to predict interaction sites in protein complexes using local composition of amino acids together with their physico-chemical characteristics. The local sequence segments (LSS) are dissected from the protein sequences using a sliding window of 21 amino acids. The list of LSSs is passed to the support vector machine (SVM) predictor, which identifies interacting residue pairs considering their inter-atom distances. We have analyzed three different model organisms of Escherichia coli, Saccharomyces Cerevisiae and Homo sapiens, where the numbers of considered hetero-complexes are equal to 40, 123 and 33 respectively. Moreover, the unified multi-organism PPI meta-predictor is also developed under the current work by combining the training databases of above organisms. The PPIcons interface residues prediction method is measured by the area under ROC curve (AUC) equal to 0.82, 0.75, 0.72 and 0.76 for the aforementioned organisms and the meta-predictor respectively. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00894-013-1886-9) contains supplementary material, which is available to authorized users

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites

<p>Abstract</p> <p>Background</p> <p>Utilization of alternative initiation sites for protein translation directed by non-AUG codons in mammalian mRNAs is observed with increasing frequency. Alternative initiation sites are utilized for the synthesis of important regulatory proteins that control distinct biological functions. It is, therefore, of high significance to define the parameters that allow accurate bioinformatic prediction of alternative translation initiation sites (aTIS). This study has investigated 5'-UTR regions of mRNAs to define consensus sequence properties and structural features that allow identification of alternative initiation sites for protein translation.</p> <p>Results</p> <p>Bioinformatic evaluation of 5'-UTR sequences of mammalian mRNAs was conducted for classification and identification of alternative translation initiation sites for a group of mRNA sequences that have been experimentally demonstrated to utilize alternative non-AUG initiation sites for protein translation. These are represented by the codons CUG, GUG, UUG, AUA, and ACG for aTIS. The first phase of this bioinformatic analysis implements a classification tree that evaluated 5'-UTRs for unique consensus sequence features near the initiation codon, characteristics of 5'-UTR nucleotide sequences, and secondary structural features in a decision tree that categorizes mRNAs into those with potential aTIS, and those without. The second phase addresses identification of the aTIS codon and its location. Critical parameters of 5'-UTRs were assessed by an Artificial Neural Network (ANN) for identification of the aTIS codon and its location. ANNs have previously been used for the purpose of AUG start site prediction and are applicable in complex. ANN analyses demonstrated that multiple properties were required for predicting aTIS codons; these properties included unique consensus nucleotide sequences at positions -7 and -6 combined with positions -3 and +4, 5'-UTR length, ORF length, predicted secondary structures, free energy features, upstream AUGs, and G/C ratio. Importantly, combined results of the classification tree and the ANN analyses provided highly accurate bioinformatic predictions of alternative translation initiation sites.</p> <p>Conclusion</p> <p>This study has defined the unique properties of 5'-UTR sequences of mRNAs for successful bioinformatic prediction of alternative initiation sites utilized in protein translation. The ability to define aTIS through the described bioinformatic analyses can be of high importance for genomic analyses to provide full predictions of translated mammalian and human gene products required for cellular functions in health and disease.</p

Insights into a dinoflagellate genome through expressed sequence tag analysis

BACKGROUND: Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging. RESULTS: Approximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair. CONCLUSION: This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production

Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Sharing Mitochondrial Clients

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.Comment: This is a reformatted version from the recent official publication in PLoS ONE (2015). This version differs substantially from first three arXiV versions. This version uses a fixed-width font for DNA sequences as was done in the earlier arXiv versions but which is missing in the official PLoS ONE publication. The title has also been shortened slightly from the official publicatio

What's new about CNBP? Divergent functions and activities for a conserved nucleic acid binding protein

Background: Cellular nucleic acid binding protein (CNBP) is a conserved single-stranded nucleic acid binding protein present in most eukaryotes, but not in plants. Expansions in the CNBP gene cause myotonic dystrophy type 2. Initially reported as a transcriptional regulator, CNBP was then also identified acting as a translational regulator. Scope of review: The focus of this review was to link the CNBP structural features and newly reported biochemical activities with the recently described biological functions, in the context of its pathological significance. Major conclusions: Several post-translational modifications affect CNBP subcellular localization and activity. CNBP participates in the transcriptional and translational regulation of a wide range of genes by remodeling single-stranded nucleic acid secondary structures and/or by modulating the activity of trans-acting factors. CNBP is required for proper neural crest and heart development, and plays a role in cell proliferation control. Besides, CNBP has been linked with neurodegenerative, inflammatory, and congenital diseases, as well as with tumor processes. General significance: This review provides an insight into the growing functions of CNBP in cell biology. A unique and robust mechanistic or biochemical connection among these roles has yet not been elucidated. However, the ability of CNBP to dynamically integrate signaling pathways and to act as nucleic acid chaperone may explain most of the roles and functions identified so far.Fil: Armas, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Coux, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Weiner, Andrea Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Calcaterra, Nora Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Evolutionary analysis of the lysine-rich N-terminal cytoplasmic domains of the gastric H+,K+-ATPase and the Na+,K+-ATPase

The catalytic α-subunits of both the Na+,K+-ATPase and the gastric H+,K+-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H+,K+-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilize particular enzyme conformations via the increased entropy it produces.Australian Research Counci

Glicoperfil do CD44: estabelecimento molecular de alvos terapêuticos no cancro da bexiga

Mestrado em Bioquímica - Bioquímica ClínicaO cancro da bexiga (BC) apresenta uma das maiores taxas de recorrência entre os tumoures sólidos e é a segunda causa de morte, relativamente a doenças do trato geniturinário. A introdução de modelos moleculares para um melhor prognóstico e desenvolvimento de terapias dirigidas efetivas, continua a ser um aspeto desafiador devido à significativa heterogeneidade molecular inter e intratumoral. No entanto, a CD44, uma proteína de membrana fortemente O-glicosilada e envolvida nas interações célula-célula, adesão celular e migração, parece desempenhar um papel crítico na progressão e disseminação do cancro da bexiga, abrindo portas para potenciais terapêuticas dirigidas. No entanto, o gene que codifica esta proteína geralmente sofre splicing alterativo, o que resulta em diversas isoformas funcionalmente distintas, de pesos moleculares variáveis e com vários locais de glicosilação. No entanto, a natureza dessas isoformas no contexto do cancro da bexiga ainda não está bem esclarecida. Com base nestas ideias, este trabalho tem como objetivo determinar as isoformas da CD44 mais clinicamente relevantes e com potencial de direcionar para clones mais agressivos. É dado particular ênfase à identificação de O-glicanos associados ao cancro, que visam aumentar o entendimento molecular para o desenho de ligandos altamente específicos. Consequentemente observou-se que a CD44 está aumentada na urina de doentes com cancro de bexiga, comparativamente com urinas controlo de indivíduos saudáveis. Esse efeito é mais pronunciado em estadios avançados da doença, particularmente após a invasão muscular, o mesmo se verifica com expressão da CD44 nos tumores de bexiga. Além disso, uma abordagem direcionada por RT-PCR demonstrou que o modelo celular de tumores superficiais de cancro da bexiga, a linha celular 5637 e os tumores de bexiga não invasivos sobre-expressam isoformas da CD44 de alto peso molecular (CD44v3-10high, CD44v8-10high, CD44slow). Por outro lado, as linhas celulares T24 e HT1376 derivadas de tumores musculo-invasivos e estes mesmos tumores sobre-expressam predominantemente CD44s, uma isoforma de menor peso molecular (CD44v3-10low, CD44v8-10low, CD44shigh). Além disso, os clones quimiorresistentes das células T24, tratadas com cisplatina, também sobre-expressam CD44s. Da mesma forma, os tumores invasores apresentaram um fenótipo semelhante, apoiando a associação da CD44 com fenótipos mais agressivos. Os estudos de glicómica e glicoproteómica envolvendo a linha celular T24 demonstraram ainda a expressão de CD44 glicosilado com antigénios sialil-Tn (CD44-STn) e di-sialil-T (dST), anteriormente associados a um pior prognóstico. Em paralelo, ensaios de imuno-histoquímica e de ligação de proximidade in situ confirmaram a existência de CD44-STn e CD44-dST em tumores musculo-invasivos. Em conclusão, CD44s, possivelmente modificada com STn e dST, tem o potencial de direcionar selectivamente para células mais agressivas de tumores de bexiga e clones quimiorresistentes, estabelecendo assim as bases moleculares para o desenho de ligandos. Estudos futuros devem-se concentrar em avaliar o impacto funcional da remodelação da CD44 para isoformas de menor peso molecular, acompanhando a transição de tumores superficiais para invasores.Bladder Cancer (BC) presents one of the highest recurrence rates amongst solid tumours, and constitutes the second deadliest disease of the genitourinary track. The introduction of molecular models for disease management and effective targeted therapeutics remains a challenging aspect due to significant inter and intra-tumour molecular heterogeneity. Nevertheless, CD44, a heavily O-glycosylated membrane protein involved in cell-cell interactions, cell adhesion and migration has been suggested to play a critical role in bladder cancer progression and dissemination, holding potential for targeted therapeutics. However, the gene encoding for CD44 generally undergoes significant alterative splicing, which results in many functionally distinct isoforms of variable molecular weights and glycosylation sites. Nevertheless, the nature of these isoforms in bladder cancer are yet to be fully disclosed. Building on these insights, this work aims to highlight clinically relevant CD44 isoforms with potential for targeting more aggressive clones. Particular emphasis is also given to the identification of cancer-associated O-glycans envisaging the molecular rational for designing highly specific cancer ligands. Accordingly, it was observed that CD44 is increased in the urine of bladder cancer patients in relation to healthy controls. This effect is more pronounced for advanced stages of the disease, particularly upon muscle invasion, mimicking CD44 expression in bladder tumours. Moreover, a targeted approach by RT-PCR demonstrated that superficial bladder cancer cell model 5637 and non-invasive bladder tumours overexpress high molecular weight CD44 isoforms (CD44v3-10high, CD44v8-10high, CD44slow phenotype). Conversely, T24 and HT1376 cell lines derived from muscle invasive tumours and invasive lesions predominantly overexpress lower molecular weight isoform CD44s (CD44v3-10low, CD44v8-10low, CD44shigh phenotype). In addition, chemoresistant clones from T24 cells challenged with cisplatin also overexpressed CD44s. Likewise, bladder tumours from patients with invasive tumours presented a similar phenotype, supporting CD44s association with more aggressive phenotypes. Glycomics and glycoproteomics studies involving T24 cell line further demonstrated the expression of CD44 glycosylated with sialyl-Tn (CD44-STn) and di-sialyl-T (dST) antigens, previously associated with poor prognosis. In parallel, immunohistochemistry and in situ proximity ligation assays confirmed the existence of CD44-STn and CD44-dST in muscle invasive tumours. In conclusion, CD44s, possibly modified with cancer-associated STn and dST glycans, holds potential to selectively target more aggressive bladder cancer lesions and chemoresistant clones, setting the molecular rational for ligands design. Future studies should now focus on disclosing the functional impact of CD44 remodelling towards lower molecular weight isoforms, accompanying transition from superficial to invasive lesions