Nuclear precursor of mature messenger RNA (pre-mRNA) splicing is one of the most highly regulated processes in eukaryotic organisms. In addition to its role in the removal of constitutive or alternative introns present in the pre-mRNA, splicing is also highly integrated into other layers of gene expression. This study investigates the potential role of the nuclear branchpoint binding protein (BBP) outside of the pre-mRNA splicing cycle. More specifically, we were interested in the biological relevance of its association with two cytoplasmic proteins Smy2 and Syh1. Smy2 and Syh1 belong to the GYF family of poly-proline binding proteins, and their roles in cell biology have not been well elucidated. Here we report that Smy2 and Syh1 act redundantly in: (i) limiting pre-mRNA accumulation when yeast cultures reach high cell density, potentially through promoting pre-mRNA decay in the cytoplasm; (ii) restricting Ty1 retrotransposition, apparently by limiting the Ty1 transcript abundance; (iii) limiting the accumulation of BBP-associated yet intronless TDA1 mRNA. With the presence of UACUAAC motif and BBP association as common features of these Smy2/Syh1 sensitive substrates, we tested if BBP interaction is required for Smy2/Syh1 function in RNA metabolism. Interestingly, we found that deletion of BBP C-terminal region (bbp∆C), which largely reduces or abolishes its association with Smy2, does not lead to similar phenotypes as observed in smy2∆ syh1∆ deletion mutant cells. In addition, mutagenesis of the TACTAAC BBP-binding site within the TDA1 coding region does not seem to affect TDA1 mRNA abundance or its sensitivity to the smy2∆ syh1∆ deletions. Therefore, we concluded that while the two BBP-binding proteins Smy2 and Syh1 impact the levels of certain cellular RNAs, this phenomenon is not strictly dependent upon BBP-Smy2 interaction and may be independent of BBP contribution. A model is proposed for Smy2 and Syh1 function in RNA metabolism based on our observations and interactions between these proteins with other factors implicated in RNA stability or translation.

This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

The eIF4E is an important eukaryotic translation initiation factor, because of its ability to bind cap at 5'end of mRNA. There are three members of this protein family found in humans: eIF4E1, eIF4E2 and eIF4E3. eIF4E1 also plays role in in export of some mRNA from nucleus to cytoplasm. This protein is mostly regulated by mTOR signaling pathway and malfunctions in regulation leads to increased cell proliferation and thus tumorogenesis. eIF4E2 plays a role in regulating of translation during embryogenesis and it is known to mediate translation in terms of hypoxia. Role of eIF4E3 is so far shrouded in mystery. Some studies suggest it might be able to suppress tumor growth, but no studies have been done on human eIF4E3. Big potential of our work is, that all proteins we work with, are human. Based on our results, the endogenous amount of eIF4E3 protein is higher than it was thought. This is one of the reasons, why this protein should not escape our attention. In my diploma thesis, I have studied physiological characteristics of cell cultures overexpressing eIF4E proteins after mTOR inhibition treatment. I have realized that the most efficient inhibitor in all tested cell cultures is PP-242, which binds directly into active site of mTOR kinase. I have cloned 3xC FLAG tagged eIF4Es constructs and used...

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Der Ribonukleinsäure (RNS) Stoffwechsel umfasst verschiedene Schritte, beginnend mit der Transkription der RNS über die Translation bis zum RNA Abbau. Poly(A) Schwänze befinden sich am Ende der meisten der Boten-RNS, schützen die RNA vor Abbau und stimulieren Translation. Die Deadenylierung von Poly(A) Schwänzen limitiert den Abbau von RNS. Bisher wurde RNS Abbau meist im Kontext von cytoplasmatischen Prozessen untersucht, ob und wie RNS Deadenylierung und Abbau in Nukleus erfolgen ist bisher unklar. Es wurde daher eine neue Methode zur genomweiten Bestimmung von Poly(A) Schwanzlänge entwickelt, welche FLAM-Seq genannt wurde. FLAM-Seq wurde verwendet um Zelllinien, Organoide und C. elegans RNS zu analysieren und es wurde eine signifikante Korrelation zwischen 3’-UTR und Poly(A) Länge gefunden, sowie für viele Gene ein Zusammenhang von alternativen 3‘-UTR Isoformen und Poly(A) Länge. Die Untersuchung von Poly(A) Schwänzen von nicht-gespleißten RNS Molekülen zeige, dass deren Poly(A) Schwänze eine Länge von mehr als 200 nt hatten. Die Analyse wurde durch eine Inhibition des Spleiß-Prozesses validiert. Die Verwendung von Methoden zur Markierung von RNS, welche die zeitliche Auflösung der RNS Prozessierung ermöglicht, deutete auf eine Deadenylierung der Poly(A) Schwänze schon wenige Minuten nach deren Synthesis hin. Die Analyse von subzellulären Fraktionen zeigte, dass diese initiale Deadenylierung ein Prozess im Nukleus ist. Dieser Prozess ist gen-spezifisch und Poly(A) Schwänze von bestimmten Typen von Transkripten, wie nuklearen langen nicht-kodierende RNS Molekülen waren nicht deadenyliert. Um Enzyme zu identifizieren, welche die Deadenylierung im Zellkern katalysieren, wurden verschiedene Methoden wie RNS-abbauende Cas Systeme, siRNAs oder shRNA Zelllinien verwendet. Trotz einer effizienten Reduktion der RNS Expression entsprechender Enzymkomplexe konnten keine molekularen Phänotypen identifiziert werden welche die Poly(A) Länge im Zellkern beeinflussen. / The RNA metabolism involves different steps from transcription to translation and decay of messenger RNAs (mRNAs). Most mRNAs have a poly(A) tail attached to their 3’-end, which protects them from degradation and stimulates translation. Removal of the poly(A) tail is the rate-limiting step in RNA decay controlling stability and translation. It is yet unclear if and to what extent RNA deadenylation occurs in the mammalian nucleus. A novel method for genome-wide determination of poly(A) tail length, termed FLAM-Seq, was developed to overcome current challenges in sequencing mRNAs, enabling genome-wide analysis of complete RNAs, including their poly(A) tail sequence. FLAM-Seq analysis of different model systems uncovered a strong correlation between poly(A) tail and 3’-UTR length or alternative polyadenylation. Cytosine nucleotides were further significantly enriched in poly(A) tails. Analyzing poly(A) tails of unspliced RNAs from FLAM-Seq data revealed the genome-wide synthesis of poly(A) tails with a length of more than 200 nt. This could be validated by splicing inhibition experiments which uncovered potential links between the completion of splicing and poly(A) tail shortening. Measuring RNA deadenylation kinetics using metabolic labeling experiments hinted at a rapid shortening of tails within minutes. The analysis of subcellular fractions obtained from HeLa cells and a mouse brain showed that initial deadenylation is a nuclear process. Nuclear deadenylation is gene specific and poly(A) tails of lncRNAs retained in the nucleus were not shortened. To identify enzymes responsible for nuclear deadenylation, RNA targeting Cas-systems, siRNAs and shRNA cell lines were used to different deadenylase complexes. Despite efficient mRNA knockdown, subcellular analysis of poly(A) tail length by did not yield molecular phenotypes of changing nuclear poly(A) tail length.

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