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- Ensterö, Mats, 1971-
(författare)
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The multi-faceted RNA molecule : Characterization and Function in the regulation of Gene Expression
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations. Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species. Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain. In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing. Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.
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| 2. |
- Rush, Margaret, 1977-
(författare)
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Regulation of RNA Processing in Human Papillomavirus Type 16
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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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