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The impact of splicing related constraints on exonic evolutionWu, Xianming January 2016 (has links)
Regulation of pre-mRNA splicing is a key process for most if not all eukaryotes. The process can, in the abstract, be considered as a series of trans-acting factors that interact with cis-motifs in the RNA to enable the removal of introns and joining of exons. As the cis factors need not only be the splice sites themselves, but also motifs in the exons, the splicing process has the potential to impose selective constraint on exonic sequence in addition to the normal selection on the amino acid content of the protein. To understand this more clearly, in this thesis, I mainly focus on a type of important and widely investigated cis-motifs, exonic splicing enhancers (ESEs), which bind with SR proteins to re-enforce the splice sites and so ensure splicing correctly. First, I explore splice-related cis-motif usage of the Ectocarpus genome, which is a species phylogenetically very distant from vertebrates but, like vertebrates in having abundant large introns. A deep phylogenetic conservation of exonic splice-related constraints is observed (Chapter II). Then I extend the analysis across taxa in a phylogenetically explicit framework. In this section stronger selection on exon end synonymous sites can be detected within humans when the exons are flanked by larger introns. Additionally I report evidence that reduced Ne might lead to larger introns and weakened splice sites. Thus I suggest an unusual circumstance in which selection (for cis-motifs to control error-prone splicing) might be stronger when population sizes are smaller; this is unexpected and would be a necessary complement to nearly-neutral theory (Chapter III). Third, I ask whether what we know about biases in the usage of ESEs and splicing control elements allows us to understand where in human genes pathogenic mutations tend to occur (Chapter IV). By examining the relationship between determinants of the usage of splice-associated cis-motifs and the distribution of human pathogenic SNPs, I found certain exons are vulnerable to splice disruption owing to low ESE density and a “fragile” exon model we proposed could describe and explain this phenomenon (Chapter IV). Finally I perform preliminary analysis, with a view to biotechnological optimization of transgenes, to address whether there might be such a thing as a tissue specific ESE. To this end I examine ESE usage in tissue specific genes. I find some preliminary evidence for tissue specific biased usage of certain ESEs.
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Regulation of RNA Processing in Human Papillomavirus Type 16Rush, Margaret January 2005 (has links)
<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>
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Regulation of RNA Processing in Human Papillomavirus Type 16Rush, Margaret January 2005 (has links)
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.
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BIOPHYSICAL CHARACTERIZATION OF ASF/SF2’S INTERACTION WITH SPLICE SITE A7 IN THE HIV GENOMEKochert, Brent Andrew 07 December 2012 (has links)
No description available.
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