A bioinformatic analysis identifies circadian expression of splicing factors and time-dependent alternative splicing events in the HD-MY-Z cell line

The circadian clock regulates key cellular processes and its dysregulation is associated to several pathologies including cancer. Although the transcriptional regulation of gene expression by the clock machinery is well described, the role of the clock in the regulation of post-transcriptional processes, including splicing, remains poorly understood. In the present work, we investigated the putative interplay between the circadian clock and splicing in a cancer context. For this, we applied a computational pipeline to identify oscillating genes and alternatively spliced transcripts in time-course high-throughput data sets from normal cells and tissues, and cancer cell lines. We investigated the temporal phenotype of clock-controlled genes and splicing factors, and evaluated their impact in alternative splice patterns in the Hodgkin Lymphoma cell line HD-MY-Z. Our data points to a connection between clock-controlled genes and splicing factors, which correlates with temporal alternative splicing in several genes in the HD-MY-Z cell line. These include the genes DPYD, SS18, VIPR1 and IRF4, involved in metabolism, cell cycle, apoptosis and proliferation. Our results highlight a role for the clock as a temporal regulator of alternative splicing, which may impact malignancy in this cellular model

The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen

Connexins: synthesis, post-translational modifications, and trafficking in health and disease

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease

MAP Kinases Phosphorylate SPF45 and Regulate Its Alternative Splicing Function: Insights onto Phosphorylation-Dependent and -Independent Effects in Ovarian Cancer Cells

Alternative pre-mRNA splicing increases proteome diversity and is important in the behavior of cells in health and disease. The processes that regulate alternative premRNA splicing are diverse and include regulation of splicing factors by phosphorylation. Here we report that the pre-mRNA alternative splicing factor SPF45 is a novel substrate of ERK, JNK and p38 MAP kinases in ovarian cancer cells. Using mutational analysis and phospho-specific antibodies, we demonstrate that MAP kinases phosphorylate SPF45 on Thr71 and Ser222 and that phosphorylation in cells is induced by a number of extracellular stimuli including PMA, EGF, serum, H20 2 and UV. Exon 6 of the death receptor Fas/CD95 has been shown to undergo alternative splicing in the presence of SPF45. Using a Fas minigene assay, we show that ERK2 and p38 inhibit SPF45 alternative splicing activity towards Fas, dependent upon these two phosphorylation sites. Other than Fas, no other pre-mRNA targets of SPF45 have been reported in mammalian cells. We generated SKOV3 cells stably-overexpressing wild-type SPF45 or a phosphorylation site mutant and performed an exon and gene array analysis to identify novel SPF45 splicing targets and genes whose expression is changed downstream of SPF45 splicing activity, respectively. From this analysis, 139 potential splicing targets and over 150 genes with altered expression were identified. We focus on four genes for validation with emphasis on ErbB2 and fibronectin. We show that SPF45 downregulates cellular proliferation in SKOV3 cells in a phosphorylation-dependent manner. Furthermore, we demonstrate that SPF45 regulates fibronectin alternative splicing, enhances inclusion of FN-EDIIIA region, and affects cellular adhesion to fibronectin matrix. We also assess SPF45 binding to SFl and SF3b15S, essential components of the spliceosome, as well as the impact of Thr71 and Ser222 mutations on this interaction. Finally, we determine the effect of SPF45 overexpression in SKOV3 cells on their drug resistance profile. This study provides a link between MAP kinase signaling and splicing factors and identifies the role of this interaction in regulating molecular processes in ovarian cancer cells. Additionally, it provides the basis to investigate the role of SPF45 in ovarian cancer and to produce successful therapeutic interventions targeting SPF45-mediated effects

Analyzing The Regulation, Stability And Functional Differences Between Sin3 Isoforms In Drosophila

SIN3 is a master transcriptional regulator, conserved from yeast to mammals, that acts as a scaffold protein for a histone modifying complex. In Drosophila, a single Sin3A gene is alternatively spliced to produce distinct SIN3 isoforms; SIN3 220, SIN3 190 and SIN3 187, that differ only at their C-terminus. These isoforms are differentially expressed during development. We have shown that there is an interplay between the predominant isoforms of SIN3, SIN3 220 and SIN3 187, that possibly regulates the overall level of SIN3 in the cell. Exogenous expression of SIN3 187 reduces the level of transcript and accelerates the proteasomal degradation of endogenous SIN3 220. This feedback can possibly ensure that the appropriate isoform is present during the correct developmental stage during embryogenesis. Differential expression of the SIN3 isoforms during embryo development suggests that they perform unique and specialized functions. The SIN3 proteins form distinct isoform specific complexes. SIN3 187 interacts with a single catalytic enzyme, the HDAC RPD3, while SIN3 220 interacts with two enzymes, RPD3 and the HDM dKDM5/LID. This differential interaction of SIN3 isoforms with distinct histone modifying activities may play a role in the non-redundant functions performed by SIN3. Using previously published transcriptome data, we have identified common and unique gene targets of SIN3 and LID. In Drosophila S2 cells, knockdown of LID results in an increase in the level of H3K9ac, H3K14ac and H3K27ac at genes commonly regulated by SIN3 and LID. Since LID preferentially interacts with the SIN3 220 complex, we have investigated the histone modification patterns established by the SIN3 isoform specific complexes. We have established Drosophila cultured cells which express either the SIN3 187 or the SIN3 220 complex. The SIN3 187 and SIN3 220 complexes establish distinct histone modification patterns at target genes and differentially regulate the expression of these genes. It is possible, that the differential histone modification patterns and the consequent alteration of expression of target genes contributes to the functional differences between the SIN3 isoforms. This work enhances our understanding of SIN3 isoform function and provides further insight into the molecular mechanisms of epigenetic control of gene expression by histone modifying complexes

From condition-specific interactions towards the differential complexome of proteins

While capturing the transcriptomic state of a cell is a comparably simple effort with modern sequencing techniques, mapping protein interactomes and complexomes in a sample-specific manner is currently not feasible on a large scale. To understand crucial biological processes, however, knowledge on the physical interplay between proteins can be more interesting than just their mere expression. In this thesis, we present and demonstrate four software tools that unlock the cellular wiring in a condition-specific manner and promise a deeper understanding of what happens upon cell fate transitions. PPIXpress allows to exploit the abundance of existing expression data to generate specific interactomes, which can even consider alternative splicing events when protein isoforms can be related to the presence of causative protein domain interactions of an underlying model. As an addition to this work, we developed the convenient differential analysis tool PPICompare to determine rewiring events and their causes within the inferred interaction networks between grouped samples. Furthermore, we present a new implementation of the combinatorial protein complex prediction algorithm DACO that features a significantly reduced runtime. This improvement facilitates an application of the method for a large number of samples and the resulting sample-specific complexes can ultimately be assessed quantitatively with our novel differential protein complex analysis tool CompleXChange.Das Transkriptom einer Zelle ist mit modernen Sequenzierungstechniken vergleichsweise einfach zu erfassen. Die Ermittlung von Proteininteraktionen und -komplexen wiederum ist in großem Maßstab derzeit nicht möglich. Um wichtige biologische Prozesse zu verstehen, kann das Zusammenspiel von Proteinen jedoch erheblich interessanter sein als deren reine Expression. In dieser Arbeit stellen wir vier Software-Tools vor, die es ermöglichen solche Interaktionen zustandsbezogen zu betrachten und damit ein tieferes Verständnis darüber versprechen, was in der Zelle bei Veränderungen passiert. PPIXpress ermöglicht es vorhandene Expressionsdaten zu nutzen, um die aktiven Interaktionen in einem biologischen Kontext zu ermitteln. Wenn Proteinvarianten mit Interaktionen von Proteindomänen in Verbindung gebracht werden können, kann hierbei sogar alternatives Spleißen berücksichtigen werden. Als Ergänzung dazu haben wir das komfortable Differenzialanalyse-Tool PPICompare entwickelt, welches Veränderungen des Interaktoms und deren Ursachen zwischen gruppierten Proben bestimmen kann. Darüber hinaus stellen wir eine neue Implementierung des Proteinkomplex-Vorhersagealgorithmus DACO vor, die eine deutlich reduzierte Laufzeit aufweist. Diese Verbesserung ermöglicht die Anwendung der Methode auf eine große Anzahl von Proben. Die damit bestimmten probenspezifischen Komplexe können schließlich mit unserem neuartigen Differenzialanalyse-Tool CompleXChange quantitativ bewertet werden

A bioengineering approach to investigating the possible role of fibronectin in neuroblastoma dormancy and relapse

ABSTRACT : Neuroblastoma (NB) is the most common extracranial solid tumor in children and is frequently diagnosed at a metastatic stage. Despite therapy, tumor relapse occurs for around 50% of high-risk NB patients. At relapse, these patients have a very poor prognosis and overall survival rate decreases. The mechanisms behind NB relapse are not well understood, but recent studies suggest that chemoresistant dormant cells are in part responsible for tumor recurrence. Fibronectin (Fn) is an extracellular matrix protein (ECM) shown to promote breast cancer cell dormancy in a TGFβ1-dependent manner. Fn presents three regions of alternative splicing: extra domain A (EDA), extra domain B (EDB), and type III constant segment (IIICS). The presence of EDA-containing Fn (EDA+ Fn) in the ECM generates a positive feedback loop by activating the TGFβ1 signaling pathway, which in turn promotes the expression of EDA+ Fn. Besides its role in dormancy, the activation of the TGFβ1 signaling pathway promotes the differentiation of stromal fibroblasts towards cancer-associated fibroblasts (CAFs), which are known to promote tumor progression and chemoresistance. Altogether, we hypothesized that the ECM protein fibronectin, and specifically EDA+ Fn, could play a critical role in neuroblastoma dormancy, chemoresistance, and relapse by activating the ERK signaling pathway in NB cells and the TGFβ1 signaling pathway in fibroblasts of the tumor environment. To validate our hypothesis here we propose three specific aims; (i) to demonstrate that fibronectin induces cancer cell dormancy and relapse in neuroblastoma; (ii) to elucidate the role of the EDA domain in Fninduced dormancy and relapse; (iii) to demonstrate how EDA+ Fn plays a crucial role in CAF activation, initiating an ECM-remodeling process that promotes tumor growth and progression.Máster en Biología Molecular y Biomedicin

Role of Sam68 in post-transcriptional gene regulation

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation