implications for health and disease

Many aspects of human physiology and behavior display rhythmicity with a period of approximately 24 h. Rhythmic changes are controlled by an endogenous time keeper, the circadian clock, and include sleep-wake cycles, physical and mental performance capability, blood pressure, and body temperature. Consequently, many diseases, such as metabolic, sleep, autoimmune and mental disorders and cancer, are connected to the circadian rhythm. The development of therapies that take circadian biology into account is thus a promising strategy to improve treatments of diverse disorders, ranging from allergic syndromes to cancer. Circadian alteration of body functions and behavior are, at the molecular level, controlled and mediated by widespread changes in gene expression that happen in anticipation of predictably changing requirements during the day. At the core of the molecular clockwork is a well-studied transcription-translation negative feedback loop. However, evidence is emerging that additional post-transcriptional, RNA-based mechanisms are required to maintain proper clock function. Here, we will discuss recent work implicating regulated mRNA stability, translation and alternative splicing in the control of the mammalian circadian clock, and its role in health and disease

Temperature does matter-an additional dimension in kinase inhibitor development

Kinase inhibitors are a major focus in drug development. Recent work shows that subtle temperature changes in the physiologically relevant temperature range can dramatically alter kinase activity and specificity. We argue that temperature is an essential factor that should be considered in inhibitor screening campaigns. In many cases, high-throughput screening is performed at room temperature or 30 degrees C, which may lead to many false positives and false negatives when evaluating potential inhibitors in the physiological temperature range. As one example, we discuss a new antimalaria compound that inhibits the highly temperature-sensitive kinase CLK3 (CDC2-like kinase 3) fromPlasmodium falciparum

A homozygous genome‐edited Sept2‐EGFP fibroblast cell line

Septins are a conserved, essential family of GTPases that interact with actin, microtubules, and membranes and form scaffolds and diffusion barriers in cells. Several of the 13 known mammalian septins assemble into nonpolar, multimeric complexes that can further polymerize into filamentous structures. While some GFP‐coupled septins have been described, overexpression of GFP‐tagged septins often leads to artifacts in localization and function. To overcome this ubiquitous problem, we have here generated a genome‐edited rat fibroblast cell line expressing Septin 2 (Sept2) coupled to enhanced green fluorescent protein (EGFP) from both chromosomal loci. We characterize these cells by genomic polymerase chain reaction (PCR) for genomic integration, by western blot and reverse transcriptase‐PCR for expression, by immunofluorescence and immunoprecipitation for the colocalization of septins with one another and cellular structures and for complex formation of different septins. By live cell imaging, proliferation and migration assays we investigate proper function of septins in these cells. We find that EGFP is incorporated into both chromosomal loci and only EGFP‐coupled Sept2 is expressed in homozygous cells. We find that endogenous Sept2‐EGFP exhibits expression levels, localization and incorporation into cellular septin complexes similar to the wt in these cells. The expression level of other septins is not perturbed and cell division and cell migration proceed normally. We expect our cell line to be a useful tool for the cell biology of septins, especially for quantitative biology

Bedeutung für die T-Zell-Aktivierung

Die Transmembrantyrosinphosphatase CD45 spielt eine zentrale Rolle in der Signaltransduktion durch den T-Zell-Rezeptor (TCR). Die CD45-Aktivität wird während der T-Zell-Aktivierung durch die Exklusion der alternativ gespleißten Exons 4, 5 und 6 gesteuert. Eine Fehlregulation dieses „activation induced alternative splicing“ genannten Prozesses trägt möglicherweise zur Ausbildung von Autoimmunerkrankungen bei. Die genauen Mechanismen, die zur Exklusion der alternativ gespleißten Exons führen, liegen bislang ebenso wie die Identität der beteiligten Spleißfaktoren weitgehend im Dunkeln. In der vorliegenden Arbeit konnte gezeigt werden, dass der Spleißfaktor U2AF26 die Exklusion alternativ gespleißter CD45-Exons in vitro und in vivo bewirkt. RNAInteraktions-Studien lassen eine Bindung von U2AF26 an regulatorische Sequenzen dieser Exons vermuten, was eine Erklärung für den beobachteten Effekt darstellen könnte. Es konnte außerdem gezeigt werden, dass die U2AF26-Expression während der T-Zell-Aktivierung stark ansteigt, was auf eine Funktion von U2AF26 in aktivierten TZellen in vivo hindeutet. Reguliert wird die Aktivität von U2AF26 durch die Interaktion mit dem Transkriptionsfaktor Gfi1. Durch eine direkte Interaktion der beiden Proteine war Gfi1 in der Lage, den U2AF26-vermittelten Ausschluss der alternativ gespleißten CD45-Exons teilweise aufzuheben. Da Gfi1 in Abwesenheit von U2AF26 keinen Einfluss auf das CD45-alternative Spleißen hatte, scheint Gfi1 spezifisch alternative Spleißprozesse zu regulieren, an denen U2AF26 beteiligt ist. Eine antagonistische Rolle von Gfi1 und U2AF26 im CD45-alternativen Spleißen konnte auch in zwei unabhängigen Mausmutanten demonstriert werden: Gfi1-defiziente und U2AF26-transgene Mäuse zeigen den gleichen Defekt im CD45-alternativen Spleißen, und zwar einen Verlust aller größeren sowie eine erhöhte Expression der kleinsten Isoform (CD45R0). In beiden Mausmutanten wurde ein schwerer Defekt in der TCR-Signalkaskade festgestellt, vermutlich ausgelöst durch eine verminderte CD45-Aktivität. Dieser Befund legt den Schluss nahe, dass die in Gfi1-defizienten und U2AF26-transgenen Mäusen überexprimierte CD45R0-Isoform eine geringere Aktivität als die größeren CD45-Isoformen aufweist. Durch die hier vorgestellten Experimente konnte eine neue Funktion von Gfi1 und U2AF26 in der Regulation des CD45-alternativen Spleißens etabliert werden und es wurde gezeigt, dass eine Manipulation des Gfi1:U2AF26-Verhältnisses in vivo zu einem Defekt in der CD45-Isoform-Expression und damit in der T-Zell-Aktivierung führt

In silico analysis of alternative splicing events implicated in intracellular trafficking during B-lymphocyte differentiation

There are multiple regulatory layers that control intracellular trafficking and protein secretion, ranging from transcriptional to posttranslational mechanisms. Finely regulated trafficking and secretion is especially important for lymphocytes during activation and differentiation, as the quantity of secretory cargo increases once the activated cells start to produce and secrete large amounts of cytokines, cytotoxins, or antibodies. However, how the secretory machinery dynamically adapts its efficiency and specificity in general and specifically in lymphocytes remains incompletely understood. Here we present a systematic bioinformatics analysis to address RNA-based mechanisms that control intracellular trafficking and protein secretion during B-lymphocyte activation, and differentiation, with a focus on alternative splicing. Our in silico analyses suggest that alternative splicing has a substantial impact on the dynamic adaptation of intracellular traffic and protein secretion in different B cell subtypes, pointing to another regulatory layer to the control of lymphocyte function during activation and differentiation. Furthermore, we suggest that NERF/ELF2 controls the expression of some COPII-related genes in a cell type-specific manner. In addition, T cells and B cells appear to use different adaptive strategies to adjust their secretory machineries during the generation of effector and memory cells, with antibody secreting B cell specifically increasing the expression of components of the early secretory pathway. Together, our data provide hypotheses how cell type-specific regulation of the trafficking machinery during immune cell activation and differentiation is controlled that can now be tested in wet lab experiments

Tageszeitabhängige Körpertemperaturrhythmen regulieren die Genexpression

Das alternative Spleißen ist ein dynamisch regulierter Mechanismus, der die Kodierungskapazität drastisch erhöht. Das alternative Spleißen ist in Tageszeitabhängig geregelt und hängt von Säugetierkörpertemperaturzyklen ab. Diese Beobachtung führte zu der Entdeckung einer körpertemperaturempfindlichen Kinase, die als Sensor wirkt, der kleine Temperaturänderungen (~ 1 ° C) in eine veränderte Phosphorylierung von RNA-Bindungsproteinen übersetzt, die wiederum über 1,500 Spleißereignisse steuern.Alternative splicing is a dynamically regulated mechanism that dramatically increases the genomes coding capacity. Alternative splicing is regulated in a time-of-day dependent manner and depends on mammalian body temperature cycles. This observation let to the discovery of a body temperature sensitive kinase, which acts as sensor translating small changes in temperature (∼ 1 °C) into altered phosphorylation of RNA-binding proteins, which in turn control over 1.500 splicing events

evolution, structure and function of metazoan splicing factor PRPF39

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer

Sec16 alternative splicing dynamically controls COPII transport efficiency

The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments

Splicing-accessible coding 3′UTRs control protein stability and interaction networks

Background 3′-Untranslated regions (3′UTRs) play crucial roles in mRNA metabolism, such as by controlling mRNA stability, translation efficiency, and localization. Intriguingly, in some genes the 3′UTR is longer than their coding regions, pointing to additional, unknown functions. Here, we describe a protein-coding function of 3′UTRs upon frameshift-inducing alternative splicing in more than 10% of human and mouse protein-coding genes. Results 3′UTR-encoded amino acid sequences show an enrichment of PxxP motifs and lead to interactome rewiring. Furthermore, an elevated proline content increases protein disorder and reduces protein stability, thus allowing splicing-controlled regulation of protein half-life. This could also act as a surveillance mechanism for erroneous skipping of penultimate exons resulting in transcripts that escape nonsense mediated decay. The impact of frameshift-inducing alternative splicing on disease development is emphasized by a retinitis pigmentosa-causing mutation leading to translation of a 3′UTR-encoded, proline-rich, destabilized frameshift-protein with altered protein-protein interactions. Conclusions We describe a widespread, evolutionarily conserved mechanism that enriches the mammalian proteome, controls protein expression and protein-protein interactions, and has important implications for the discovery of novel, potentially disease-relevant protein variants

Recruitment of a splicing factor to the nuclear lamina for its inactivation

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments