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Cleavage and polyadenylation of a precursor mRNA is important for transcription termination, mRNA stability, and regulation of gene expression. This process is directed by a multitude of protein factors and cis elements in the pre-mRNA sequence surrounding the cleavage and polyadenylation site. Importantly, the location of the cleavage and polyadenylation site helps define the 3’ untranslated region of a transcript, which is important for regulation by microRNAs and RNA binding proteins. Additionally, these sites have generally been poorly annotated. To identify 3’ ends, many techniques utilize an oligo-dT primer to construct deep sequencing libraries. However, this approach can lead to identification of artifactual polyadenylation sites due to internal priming in homopolymeric stretches of adenines. Previously, simple heuristic filters relying on the number of adenines in the genomic sequence downstream of a putative polyadenylation site have been used to remove these sites of internal priming. However, these simple filters may not remove all sites of internal priming and may also exclude true polyadenylation sites. Therefore, I developed a naïve Bayes classifier to identify putative sites from oligo-dT primed 3’ end deep sequencing as true or false/internally primed. Notably, this algorithm uses a combination of sequence elements to distinguish between true and false sites. Finally, the resulting algorithm is highly accurate in multiple model systems and facilitates identification of novel polyadenylation sites.

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<p>Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer.</p><p>The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition.</p><p>This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.</p>

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition. This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.

The microtubule-associated protein tau forms pathological neuronal filaments in Alzheimer's disease (AD) and other neurodegenerative disorders, known collectively as tauopathies. Previous studies in transgenic mouse models of AD suggest that reducing tau expression may be safe and beneficial for the prevention or treatment of AD and possibly other tauopathies. As a first step toward identifying novel therapeutic strategies to reduce tau levels, the studies presented in this dissertation aim to investigate the role of the human tau 3'-untranslated region (3'-UTR) in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. The exact sequence of these two 3'-UTR isoforms was determined by rapid amplification of cDNA 3'-ends (3'-RACE), and the two 3'-UTR isoforms were cloned into a luciferase reporter vector. Using these reporter constructs, the expression of these isoforms was found to be differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y by luciferase assays and quantitative PCR (qPCR). Through an unbiased screen of tau 3'-UTR deletions and fragments using luciferase reporter constructs, several regions in the long tau 3'-UTR isoform that contain regulatory cis-elements were identified. Additionally, several microRNAs were computationally identified as candidates that might bind the long tau 3'-UTR and thereby differentially control the expression of long versus short tau 3'-UTR isoforms. Screening these candidate microRNAs via luciferase reporter assay identified miR-34a, which was subsequently shown to repress the expression of endogenous tau protein and mRNA in M17D cells using Western blot and qPCR, respectively. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. Taken together, these studies suggest that the expression of the two tau 3'-UTR isoforms is differentially regulated and that this differential regulation is due to the presence of regulatory cis-elements found only in the long tau 3'-UTR isoform, including a binding site for miR-34 family members. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies.

Drought stress is considered one of the most devastating abiotic stress factors that limit crop productivity for modern agriculture worldwide. There is a large range of physiological and biochemical responses induced by drought stress. The responses range from physiological and biochemical to regulation at transcription and posttranscriptional levels. Post-transcription, the products encoded by eukaryotic genes must undergo a series of modifications to become a mature mRNA. Polyadenylation is an important one in terms of regulation. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially when different mRNAs from the same gene may be polyadenylated at more than one position. This alternative polyadenylation (APA) has numerous potential effects on gene regulation and function. I have studied the impact of drought stress on APA, testing the hypothesis that drought stress may give rise to changes in the usage of poly(A) sites generating different mRNA isoforms. The results showed that usage of poly(A) sites that lie within 5’-UTRs and coding sequence (CDS) changes more than usage of sites in other regions due to drought stress. Alternative polyadenylation is meditated by the polyadenylation complex of proteins that are conserved in eukaryotic cells. The Arabidopsis CPSF30 protein (AtCPSF30), which is an RNA-binding endonuclease subunit of the polyadenylation complex, plays an important role in controlling APA. Previous study showed that poly(A) site choice changes on a large scale in oxidative stress tolerant 6 (oxt6), a mutant lacking AtCPSF30. Within the mutant/WT genotypes, there are three classes of poly(A) site, wild type specific, oxt6 specific, and common (both in wild type and mutant). The wild type specific and oxt6 specific mRNAs make up around 70% of the total of all mRNA species. I hypothesize that the stability of these various mRNA isoforms should be different, and that this is a possible way that AtCPSF30 regulates gene expression. I tested this by assessing the influence poly(A) sites can have on the mRNA isoform’s stability in the wild type and oxt6 mutant. My results show that most mRNA isoforms show similar stability profiles in the wild-type and mutant plants. However, the mRNA isoforms derived from polyadenylation within CDS are much more stable in the mutant than the wild-type. These results implicate AtCPSF30 in the process of non-stop mRNA decay. Messenger RNA polyadenylation occurs in the nucleus, and the subunits of the polyadenylation complex that meditate this process are expected to reside within the nucleus. However, AtCPSF30 by itself localizes not only to the nucleus, but also to the cytoplasm. AtCPSF30 protein contains three predicted CCCH-type zinc finger motifs. The first CCCH motif is the primary motif that is responsible for the bulk of its RNA-binding activity. It can bind with calmodulin, but the RNA-binding activity of AtCPSF30 is inhibited by calmodulin in a calcium-dependent manner. The third CCCH motif is associated with endonuclease activity. Previous studies demonstrated that the endonuclease activity of AtCPSF30 can be inhibited by disulfide reducing agents. These published results suggest that there are proteins that interact with AtCPSF30 and act through calmodulin binding or disulfide remodeling. To test this hypothesis, I screened for proteins that interact with AtCPSF30. For this, different approaches were performed. These screens led me to two proteins-one protein that is tyrosine-phosphorylated and whose phosphorylation state is modulated in response to ABA, which well-known ABA regulates guard cell turgor via a calcium-dependent pathway, and the other is ribosome protein L35(RPL35), which plays an important role in nuclear entry, translation activity, and endoplasmic reticulum(ER) docking. These results suggest that multiple calcium-dependent signaling mechanisms may converge on AtCPSF30, and AtCPSF30 might be directly interact with ribosome protein.

<p>Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. </p><p>PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP.</p><p>Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.</p>

<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>