Molecular motions at the 5 stem-loop of U4 snRNA: Implications for U4/U6 snRNP assembly

Das humane 15.5K Protein bindet an die 5' Schleife der U4 snRNA (KtU4). Es begünstigt den Aufbau des Spliceosoms U4/U6 snRNP und ist notwendig für die Zusammenführung des 61K Proteins und des 20/60/90K Proteinkomplexes mit der U4-snRNA. In der kristallografischen Struktur des 15.5K-U4 snRNA Komplexes gehört die RNA Faltung zu der Familie der so genannten kink-turn (K-turn) Motive. Dieses Motiv ist durch einen scharfen Knick im Phosphodiesterrückgrat gekennzeichnet. Zwei Helixstämme sind durch eine purinreiche, asymmetrische, innere Schleife verbunden. Diese enthält eine herausgeklappte Uridinbase und zwei aufeinander folgende verschobene G-A Basenpaare. Der kürzere Helixstamm ist mit einer externen fünffachen Schleife verbunden. Mit Hilfe von Molekulardynamiksimulationen konnte ich zeigen, dass die Faltung von KtU4 durch die Proteinbindung unterstützt wird. Konformationsänderungen wie die Umwandlung zwischen alternativen Purin-Stapelschemata, der Verlust von G-A Basenpaaren und die Öffnung des K-turns (k-e Bewegung) trat nur in den Simulationen der ungebundenen RNA auf. Diese Simulationen zeigen zum ersten Mal die Dynamik des K-turn Motivs mit atomarer Auflösung und finden sich in hervorragender Übereinstimmung mit experimentellen Resultaten, die durch chemische Erprobung und FRET-Studien an Einzelmolekülen der RNA gesammelt wurden. In der ungebundenen RNA wurde die k-e Bewegung gleichermaßen durch den Verlust der G-A Basenpaare in der inneren Schleife und durch Flexibilität im Phosphatrückgrat der beiden Helixstämme hervorgerufen. Jedoch war der Verlust der G-A Basenpaare allein nicht ausreichend, um eine große Entfaltung der ungebundenen RNA zu erreichen. Eine Untersuchung der Eigenmodi der RNA Bewegung konnte zeigen, dass der Verlust der G-A Basenpaare nur mit dem ersten Eigenmodus korreliert ist aber nicht mit dem dritten Eigenmodus, der die k-e Bewegung beschreibt. Aufgrund dieser Ergebnisse komme ich zu dem Schluss, dass sich die G-A Basenpaare erst bei der Bindung an das 15.5K Protein bilden und d adurch selektiv die Ausrichtung der Helixstämme stabilisieren. Die externe Schleife konnte in der Kristallstruktur des 15.5K-KtU4 Komplexes nicht aufgelöst werden. In den Simulationen des Komplexes nahm sie eine bestimmte Orientierung ein, die nicht in der ungebundenen RNA beobachtet wurde. Sie trat auch dann nicht auf, wenn die natürliche Sequenz durch andere externe Schleifen ersetzt oder die Helixstämme verlängert wurden. Auf Grund der Simulationsergebnisse schlage ich vor, dass die nicht vorhandene Stapelung zwischen dem letzten Basenpaar des Helixstamms und dem benachbarten Nukleotid in der externen Schleife wichtig ist für die korrekte Faltung der RNA und eine wichtige Rolle in der nachfolgenden Bindung des 61K Proteins an die U4 snRNA spielt

Loss of G–A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif

Upon binding to the 15.5K protein, two tandem-sheared G–A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA (‘k–e motion’) were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k–e motion in Kt-U4 is triggered both by loss of G–A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G–A base pairs is correlated along the first mode but anti-correlated along the third mode with the k–e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G–A base pairs and modified stacking interactions. Thus, loss of G–A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G–A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems

DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox- Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.Link_to_subscribed_fulltex

Dissecting the role of distinct OCT4-SOX2 heterodimer configurations in pluripotency

The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.Link_to_subscribed_fulltex

The ubiquitination landscape of the influenza A virus polymerase.

During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1 and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

� 2016 The Authors. Published under the terms of the CC BY 4.0 license The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization.Link_to_subscribed_fulltex

The Relationship of Cytokines IL-13 and IL-17 with Autoantibodies Profile in Early Rheumatoid Arthritis

Aims. In the present study, we aimed to assess the concentrations of IL-13 and IL-17 in serum of patients with early rheumatoid arthritis (eRA), the investigation of correlation between the concentrations of these cytokines and disease activity score, and the concentration of some autoantibodies and the evaluation of the utility of IL-13 and -17 concentration measurements as markers of disease activity. Materials and Methods. Serum samples were collected from 30 patients and from 28 controls and analysed parameters. Results. The serum concentrations of IL-13, IL-17, anti-CCP, and IgM-RF were statistically significantly higher in patients with eRA, compared to the controls. IL-13 concentrations in the severe and moderate groups with eRA were statistically higher than in the mild and control groups. Also, in the case of IL-17, serum concentrations increased proportionally with the disease activity of eRA. We observe that concentrations of IL-13 and -17 did not correlate with autoantibodies. IL-17 concentration significantly positively correlated with CRP, while IL-13 concentration significantly negatively correlated with CRP. Disease activity score, DAS28, was strongly positively correlated with levels of ESR and weakly positively correlated with concentrations of anti-RA33 autoantibodies. IL-13 has a higher diagnostic utility than IL-17, CRP, ESR, IgM-RF, and anti-CCP as markers of disease activity. Conclusions. The presence of higher IL-13 and IL-17 serum levels in patients, compared with those of controls, confirms that these markers, found with high specificity, might be involved in the pathogenesis of eRA. IL-13 and IL-17 might be of better usefulness in the prediction of eRA activity status than IgM-RF and anti-CCP

Nerve compression due to benign tumors or ganglion cysts in the upper limb – case series

Tumor nerve compressions in the upper limb are relatively rare, usually involving ganglion cysts and benign tumors. We present a case series of five patients with peripheral nerve compression in the upper limb due to tumor or cystic masses- ulnar nerve compression in the Guyon’s tunnel due to a ganglion cyst, large median nerve schwannoma compressing anterior interosseous nerve and median nerve, voluminous lipoma compressing median nerve in the proximal forearm, superficial branch of radial nerve compression by a synovial cyst and elbow region lipoma compressing radial nerve. In the beginning, those benign lesions are asymptomatic but, as they continue to grow adjacent to a peripheral nerve clinical manifestations appear progressively as compressive neuropathies. After a preoperative imagistic analysis, tumor resection with careful dissection, in order to preserve the neurovascular structures, is the elective surgical procedure in order to obtain an optimal functional recovery

Toponymes et appellatifs géographiques

Cojocaru Vlad. Toponymes et appellatifs géographiques. In: Nouvelle revue d'onomastique, n°47-48, 2007. pp. 185-197