Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence

The phospho-regulation of Ascl2 in homeostatic stem cell maintenance and differentiation in the small intestine

Zusammenfassung Der Transkriptionsfaktor Achaete-Scute (Ascl2) aus der Familie der basic Helix-loop- Helix Proteine (bHLH) spielt eine essentielle Rolle für Stammzellen im Dünndarm des Menschen wie auch der Maus. Das Gen Ascl2 kodiert für das Protein Ascl2. Ascl2 hat Einfluss auf die eigene Aktivität durch eine Rückkopplungsschleife, die durch eine basale Aktivierung von Wnt reguliert wird und ermöglicht dadurch ein abgegrenztes Kompartiment für Stammzellen im Darmepithelium. Aus diesem Grund wird Ascl2 als Schlüsselregulator der Determinierung zwischen Proliferation und Differenzierung im Darm angesehen. Die Expression von Ascl2 ist auch in die Tumorbildung von kolorektalen Karzinomen involviert, da Ascl2 die Proliferation von malignen Zellen antreibt. Das bringt die Frage auf, welche Eigenschaften von Ascl2 zur Tumorentwicklung im Darm führen? Verwandte Proteine von Ascl2 zeigen, dass deren biologische Wirkung von deren Phosphorylierungsstatus abhängt. Deswegen wurde in dieser Arbeit folgende zentrale Frage adressiert: Welchen Einfluss hat die Regulation von Ascl2 durch Phosphorylierung auf die homöostatische Aufrechterhaltung von Stammzellen und Zelldifferenzierung im Darm? Um den Phosphorylierungsstatus von Ascl2 in vitro zu bestimmen, sowie herauszufinden welche Cyclin-abhängigen Kinasen daran beteiligt sind und wie sich dies auf die Proteinstabilität von Ascl2 auswirkt, wurden biochemische Experimente durchgeführt. Um die Effekte von Ascl2 Phosphorylierung zu bestimmen, wurden Darmorganoide der Maus mit einem Doxycyclin-induzierbaren Vektorsystem transferiert, welches die Expression von Wildtyp-Ascl2 beziehungsweise eine nicht- phosphorylierbare Mutante erlaubt. Die Resultate zeigen, dass Ascl2 hauptsächlich von der Kinase Cdk1/CyclinB phosphoryliert wird und die Inhibierung Cyclin-abhängiger Ascl2 Phosphorylierung sich positive auf Stabilität und Abundanz des Proteins auswirkt. Es konnte gezeigt werden, dass inhibierte Phosphorylierung von Ascl2 zu verschlechtertem Wachstum von Darmorganoiden führt und sogar Abwehrmechanismen auslöst, um die Expression von dephosphoryliertem Ascl2 zu supprimieren. Diese Untersuchungen liefern erste Einblicke in die Rolle von phosphoryliertem Ascl2 bei der Aufrechterhaltung eines Stammzellekompartiments im Dünndarm.The achaete-scute family bHLH transcription factor 2 (Ascl2) is essential for stem cells in the human as well as the mouse small intestine. The gene Ascl2 is coding for the protein Ascl2. Ascl2 was found to have the ability to self-activate its expression through a feedback loop, which is activated by basal Wnt signalling. By that Ascl2 is capable of maintaining a discrete compartment of stem cells in the intestinal epithelium. Therefore Ascl2 can be considered as a key force of cell fate regulation in the intestine. Ascl2 is known to be involved in colorectal cancer formation by maintaining a proliferative stem cell character in malignant cells. This raises the question what features of Ascl2 expression lead to the formation of malignancies in the intestine? Related genes of Ascl2 showed that phosphorylation is crucial for their fundamental fate-driving forces in cells. Hence, the role of Ascl2s phosphorylation status became focus of interest: What impact does the phospho-regulation of Ascl2 have on homeostatic stem cell maintenance and differentiation in the small intestine? To examine the role of cyclin-dependent phosphorylated Ascl2 as well as dephosphorylated Ascl2, a human colorectal cancer cell line and intestinal organoids from mice were analysed. Biochemical experiments were conducted to determine the phosphorylation status of Ascl2 in vitro, identifying which cyclin-dependent kinases are involved and to obtain data on phosphorylation-depending protein stability. Organoids were transduced with a tetracycline-inducible vector system, which allows the expression of wild-type Ascl2 and a non-phosphorylatable mutant, respectively, to assess the effects of phosphorylation of Ascl2. The results demonstrate that Ascl2 is mainly phosphorylated by cyclin-dependent kinase Cdk1/CyclinB. The prevention of cyclin-dependent phosphorylation of Ascl2 increases its stability and abundance exceedingly. Prevention of phosphorylation of Ascl2 in intestinal organoids leads to impaired growth and subsequently causes defence mechanisms in those organoids trying to suppress the expression of dephosphorylated Ascl2. This study provides first insights into the role of phosphorylation of Ascl2 in small intestine stem cell maintenance.submitted by Clara Duré, BScZusammenfassung in deutscher SpracheUniversität Innsbruck, Masterarbeit, 2018(VLID)287807

Immune response of Staphylococcus aureus strains in a mouse mastitis model is linked to adaptive capacity and genotypic profiles

Staphylococcus aureus is one of the most frequently isolated major pathogens from intramammary infections (IMI) worldwide. The mechanisms by which S. aureus IMI are established and maintained in dairy cows involve both bacterial escape strategies and modulation of the host immune response. Moreover, it was shown that different S. aureus strains have varying effects on the immune response. The aim of this study was to investigate the immune response in a mouse mastitis model of two S. aureus strains isolated from bovine IMI with different clinical manifestation (persistent-P or non-persistent-NP), phenotypic and genotypic profile. Both strains were capable of establishing an IMI after 264 h post inoculation (pi). Strain A (NP) showed a more aggressive behaviour than strain B (P) at early stages of IMI, while strain B multiplied initially at a lower rate but increased its replication capacity from 120 h pi to the end of the study (264 h pi). Strain A triggered a stronger initial inflammatory response compared with strain B inducing higher gene and protein expression of TLR2, NF-κB activation and higher gene expression of IL-1α at initial stage of IMI (6?12 h pi) but inducing extensive mammary tissue damage. Immune cells response was different for each S. aureus strain throughout the course of infection, showing mammary glands inoculated with strain A greater initial immune cells stimulation compared with strain B and then a second immune cells stimulation (from 120 to 264 h pi) represented by monocytes-macrophages, T and B lymphocytes, mainly stimulated by strain B, consistent with inflammatory process becoming chronic. Strain-specific pathogenicity observed underscores the importance of pathogen factors in the progression of the infectious process. These results contribute to increase the available information on host-pathogen interaction and point out for the need of further research to expand the knowledge about these interactions for developing new strategies to intervene in the IMI progress.Fil: Pereyra, Elizabet Amanda Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Sacco, Sofía Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Duré, Andrea Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Baravalle, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Renna, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Andreotti, Carolina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Monecke, Stefan. Technische Universität Dresden; Alemania. Alere Technologies GmbH; AlemaniaFil: Calvinho, Luis Fernando. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Clínicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Dallard, Bibiana Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentin