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<p>High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. </p><p>Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus.</p><p>In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. </p><p>Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.</p>

<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>

High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus. In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.

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.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.

La RNase P est une endoribonucléase responsable de la maturation de l’extrémité 5’ des ARNt prématures. Holoenzyme très conservée, elle est constituée d’une composante ARN formant le noyau catalytique et d’une composante protéique dont le nombre de sous-unités est variable : une protéine chez les bactéries, 5 chez les archées et d’au moins 9 chez les eucaryotes. Les eucaryotes possèdent également une autre endoribonucléase, la RNase MRP dont la composition est proche de la RNase P tant au niveau ribonucléique que protéique mais avec une spécificité de substrat propre. Dans cette étude, nous proposons une méthode originale et spécifique pour purifier la RNase P et la RNase MRP de S. cerevisiae. Grâce à la microscopie électronique et au traitement d’images, nous avons déterminé la première structure de ces deux holoenzymes à une résolution d’environ 1.5 nm. Ces structures révèlent une architecture modulaire commune où les protéines stabilisent la composante ARN et contribuent à l’édification de cavités et de conduits. Les spécificités structurales sont localisées en des positions stratégiques pour l’identification et la coordination du substrat. / Ribonuclease P (RNase P) is an endoribonuclease that cleaves the 5'-leader sequence of pre-tRNAs. RNase P is conserved between all taxonomic kingdoms and consists of a catalytic RNA subunit and protein components of variable size, from one protein in bacteria to 5 proteins in archae and at least 9 proteins in eukaryotic cells. In addition to RNase P, eukaryotes possess the RNase MRP which has a related RNA core and shares 8 proteins subunits with RNase P but with its own substrate specificity. Here, we propose an original method to purify specifically RNase P and RNase MRP from S. cerevisiae. Using electron microscopy and image processing, we solved the first structure of these two holoenzymes at a resolution of about 1.5 nm. We showed that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules.Structural features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence.

The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.