Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren. / How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals.

Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease. / text

Giardia intestinalis is an anaerobic protozoan pathogen, agent of the disease known as giardiasis. The regulation of gene expression during giardia cell- and life-cycle has been poorly studied so far, with the exception of variable surface proteins, which constitute the immunoprotective coat of the cell. In this diploma thesis, we focus on the possible role of the 3' untranslated region (3'UTR) of mRNA that mediate stability and localization of mRNA transcripts. We use RNA binding proteins of PUF family, which control the function of the target transcripts by their repression, activation or sequestration, to monitor and verify the role of 3'UTRs. These only eukaryotic proteins are highly evolutionarily conserved. Each of them contain highly conserved C-terminal domain, which specificly binds to 3'UTR of mRNAs. We have identified five different PUF proteins in the genome of G. intestinalis (GiPUF), cinfirmed their expression in G. intestinalis trophozoites and located all five proteins in the cytoplasm. GiPUF2, GiPUF3 and GiPUF5 show an additional affinity to the surface of the endoplasmic reticulum. We have identified the C-terminal binding domain in protein sequences of all GiPUF. The most conserved GiPUF4 contain eight binding sites, nearly identical to the binding site of human Pum1 protein,...

<p>Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. </p><p>Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids.</p><p>The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. </p><p>In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.</p>

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids. The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.

<p>SR proteins are a family of splicing factors involved in the regulation of both constitutive and alternative splicing of pre-mRNAs. Despite years of studies, several big questions still remain: how the expression levels of SR proteins are regulated; what are the underlying mechanisms responsible for SR proteins-mediated gene regulation; what are the physiological targets of SR proteins in vivo. In my dissertation study, I am focusing on two members of the family, SF2/ASF and SRp20, to study their functional involvement in regulating microRNA/mRNA biogenesis and their own expression. </p><p>Negative feedback regulation is a common mechanism maintaining the steady-state level of SR proteins (i.e. SC35 and SRp20), and several mechanism may be involved. In order to test if miRNAs are also involved in such negative feedbacks, small RNA sequencing was used to identify differentially expressed miRNAs after SF2/ASF overexpression in an inducible stable cell line system. Among the 40 differentially expressed miRNAs, miR-7 is particularly interesting, because it is also predicted to target SF2/ASF, which forms a negative feedback regulation. This is indeed the case as shown by luciferase reporter assay and overexpression/knocking down of miR-7 in vivo. To our knowledge, this is the first identified negative feedback circuit between a SR protein and a miRNA, which may be a general mechanism in regulating SR protein homeostasis.</p><p>To characterize the mechanism underlying SF2/ASF-enhanced miRNA biogenesis, I have employed a series of molecular and biochemical approaches to pinpoint the key molecular interactions in a minigene system, which is consist of miR-7 embedded intron and the flanking exons of its host gene. By manipulating the splicing pattern of such minigene, I have uncovered a splicing-independent function of SF2/ASF in regulating miRNA biogenesis. Directly binding between SF2/ASF protein and pri-miR-7 was demonstrated by Cross-linking and immunoprecipitation assay (CLIP) and RNA affinity purification. The precise binding site was then pinpointed by combining computational prediction and mutagenesis assay. Finally, by using in vitro pri-miRNA processing assay, I showed that SF2/ASF can promote the Drosha cleavage step of pri-miR-7 through directly association with the predicted binding site. So far, this is the first SR protein discovered, which is directly involved in miRNA biogenesis. Moreover, our preliminary data also suggested that SF2/ASF may promote miRNA biogenesis in other steps after Drosha cleavage; and different SR proteins can regulate miRNA biogenesis in a substrate-specific manner. Taken together, SR family of splicing factors may be broadly involved in miRNA biogenesis through direct interactions.</p><p>In order to study the general involvement of SR proteins in RNA biogenesis, one important step stone is to have a better profile of their targets in vivo. To achieve this, I focused on SRp20, another classic SR protein. Photoactivatable-Ribonucleoside-Enhanced Cross-linking and immunoprecipitation assay combined with deep sequencing (PAR-CLIP-seq) was used to identify the binding partners of SRp20 globally, which is subsidized by candidate gene validations. Consistent with the literature, I found that SRp20 primarily targets exonic regions for splicing regulation, and such interactions are likely to be sequence dependent on the CWWCW motif. Surprisingly, I also observed extensive binding between SRp20 and the 3' UTRs of mRNA, which may affect the choice of alternative polyadenylation sites. The underlying mechanisms are being investigated by a variety of molecular methods. </p><p>In summary, I have identified a subset of miRNAs, the expression of which can be regulated by SF2/ASF in a splicing independent manner. This is the first SR protein identified in regulating miRNA biogenesis. One of the upregulated miRNAs, miRNA-7 can form a negative feedback with SF2/ASF by negatively regulating the expression of SF2/ASF on translational level. By using PAR-CLIP method, I have identified the genome-wide binding partners of SRp20 in vivo. When SRp20 binds to the exonic regions, it potentially affects the alternative splicing patterns of nearby introns. Interestingly, the 3' end choices for a subset of genes may be regulated by SRp20 through directly binding, which may be a new mechanism for the regulation of 3' end processing.</p> / Dissertation

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.

RNA localization, a regulated step of gene expression, is fundamentally important in development and differentiation. In multidisciplinary experiments, we discovered that RNA (mis)localization underlies the human disease myotonic dystrophy (DM). DM, the most prevalent adult muscular dystrophy, is caused independently by two alleles: DM1 is characterized by a (CTG)n expansion in the DM kinase (DMPK) gene 3' untranslated region while DM2 has a mutation in a small presumptive RNA binding protein. These analyses were guided by disease characteristics and have provided insights to DM's cytopathology, cell biology and molecular genetics. Examining muscle biopsies, it is demonstrated here that DM kinase mRNA is specifically subcellularly localized within normal human muscle and that DM kinase mRNA harboring the 3’UTR mutation (DM1) is mislocalized in DM patient muscle to cytoplasmic areas characteristic of DM disease pathology. Thus, the disease mutation alters the cellular distribution of the effected message. DMPK mRNA mislocalization causes altered DM kinase protein localization, correlates with novel phosphoprotein appearance and can account for DM’s diseased phenotype. While we were fortunate to access DM patient tissue to establish these key findings, the system does not lend itself to experimental manipulation. Hence, I established a disease- relevant tissue culture system, which recapitulates DMPK trafficking, Employing this system; I elucidate a complementary role for the DM2 gene product as a localization factor for DMPK mRNA (DM1 gene product). Comprehensive RNA-protein interaction experiments reveal the DM2 protein specifically and selectively recognizes a small, definitive area within the DMPK RNA 3'UTR. Detailed biochemical, cytological and functional experiments reveal 1) the DM2 protein colocalizes with DMPK mRNA, 2) the small area of the DMPK 3’UTR bound by pDM2 acts to properly localize a reporter construct and 3) disruption of the DM2 protein results in DMPK mRNA mislocalization. These data establish mRNA localization as a vital process underlying human disease etiology. Moreover, they reveal DM1 and DM2 gene products function in the same molecular pathway and that mutation of either causes DMPK mRNA mislocalization, leading to disease. These data have apparent application to several neuromuscular disorders and open a plethora of novel research avenues, both basic and applied. / text

Black grouse Tetrao tetrix is a bird species composed of large, continuous as well as severely reduced and fragmented populations, making it an optimal species to investigate how genetic diversity is affected by habitat fragmentation. I have focused on genetic diversity in the Major Histocompatibility Complex (MHC) to measure the ability of the black grouse to respond to environmental changes. I partly characterized MHC class II in black grouse and found striking similarities with chicken MHC class II. I demonstrated that black grouse possess a similar compact MHC as chicken with few MHC class II B (BLB) and Y (YLB) loci. I did not find evidence of balancing selection in YLB so I concentrated further studies on BLB. I developed a PCR-based screening method for amplifying and separating expressed BLB alleles in European black grouse populations. Small fragmented populations had lost neutral genetic diversity (based on microsatellites and SNPs) compared to samples from the historical distribution and contemporary large populations. There was also a trend, albeit less pronounced, for reduced MHC diversity in these populations. Neutral markers in small isolated populations were affected by increased levels of genetic drift and were therefore genetically differentiated compared to other populations. MHC markers on the other hand, were not subjected to genetic drift to the same extent probably due a long historic process of balancing selection. Inferences of heterozygosity and evolutionary patterns as well as detailed correlations to reproductive success and diseases cannot be performed until MHC can be amplified in a locus-specific manner. Therefore, I developed a single locus sequence-based typing method for independently amplifying MHC class II B loci (BLB1 and BLB2). I found that BLB1 and BLB2 were duplicated in a common ancestor to chickens and black grouse and that these loci are subjected to homogenizing concerted evolution due to inter-genetic exchange between loci after species divergence.  I could also verify that both BLB1 and BLB2 were transcribed in black grouse and under balancing selection. This collection of work has significance for future conservation of black grouse as well as research and management of zoonotic diseases.

The purpose of this project was to identify and evaluate nucleotide sequences enhancing translation of proteins in Chinese hamster ovary (CHO) cells. Candidate sequences were placed in the 5´-untranslated region (UTR) or 3´ UTR respectively and evaluated in a CHO-based expression system with a fluorescent Fc-fusion protein as a model protein.Five plasmid vectors were constructed, two of which designed to have a randomized nucleotide library in their 5´ and 3´ UTR respectively, and three of which designed to hold varying repeats of a known enhancing translation (ET) sequence in their 5´ or 3´ UTR. The plasmid constructs were transfected into CHO cells and the protein expression was analyzed both by fluorescence intensity in single cells using flow cytometry and in bulk by monoclonal antibody titer analysis based on Protein A affinity.The main result is that both flow cytometry and titer analysis indicate that insertion of five repeats of the ET in the 5´UTR has a negative effect on protein expression as compared to the control which had no ET repeats. Results related to the insertion of three ETs in the 5´ UTR were ambiguous. The titer analysis indicated that it had a negative effect on the protein expression compared to the control which had no ET repeats, whereas the flow cytometry results suggest that the effect is negligible. Transfection of library plasmids was unsuccessful; hence no library expression analysis results were achieved. Due to the time constraints of the project, the reason for the unsuccessful transfection of library plasmids was not investigated, but the LTX transfection method is stated as a highly plausible cause.Based on the outcome of this study, two recommendations for future work are suggested. The first one is to continue the focus on UTR sequences in terms of library screening, and to improve the method of transfecting library plasmid constructs into CHO cells using lipofection. The second suggestion for further studies is to test different UTR sequence lengths without involving potential ETs, to rule out the effect and positions of the ETs and investigate the expressional effect of UTR length solely.