Alternative Splicing and mRNA Stability: Control of SERCA2 Expression
| Autor: | Misquitta, Christine M. |
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| Rok vydání: | 2009 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | The overall hypothesis is that sequences in the 3'-region and tissue-specific protein factos contribute to the stability of the sarco/endoplasmic reticulum (SERCA) pump mRNA. Calcium pumps, which sequester calcium ions into the sacroplasmic reticulum in left venticular myocytes (LVM) or stomach smooth muscle cells (SSM), are encoded by the SERCA2 gene. SERCA2 transcripts are alternatively spliced at base 3495, giving mRNA which differ mainly in the 3'-UTR and also encode two proteins (SERCA2a in LVM and SERCA2b in SSM) with small differences in their C-termini. The rate of SERCA2 transcription in LVM and SSM is similar, yet there is 20 to 30-fold more SERCA2 mRNA and 100-fold more SERCA2 protein in LVM than SSM. This thesis relates the differences in the SERCA2 expression level in the two tissues to their 3'-domains via sequences affecting mRNA stability. Using primary cell cultures of LVM and SSM, transcription was inhibited and the half life of SERCA2 mRNA was determined. The half-life of SERCA2 mRNA in LVM was 27 ± 3 h while in SSM it was 13 ± 0.5 h. Thus, SERCA2 mRNA is more stable in LVM than in SSM. The abundance of SERCA2 and control RNA in isolated nuclear and cytoplasmic fractions was compared to determine whether the difference in the SERCA2 mRNA abundance originates in the nucleus or the cytoplasm. SERCA2a is already 20-fold more abundant than SERCA2b in the nucleus suggesting a nuclear mechanism of decay or differences in export rates. Since SERCA2a and 2b mRNA differ in their 3'regions, the decay rates of the 3'-region mRNA (starting from 3446) from SERCA2a and 2b was examined in nuclear and cytoplasmic extracts of both tissues. In all extracts, except SSM cytoplasm, the 3'-region mRNA of SERCA2a was significantly more stable than that of SERCA 2b with the biggest difference being oberseved in LVM nuclear extracts. In vitro decay experiements using poly A+ RNA isolated from the two tissues also showed that, in the presence of LVM nuclear protein extracts, the SERCA2a mRNA was more stable than the SERCA2b. Thus cis-acting elements (RNA sequences) in the 3'-region may play a role in SERCA2 mRNA stability. To identify possible cis-acting elements of SERCA2a and 2b, individual decay rates of large overlapping fragments that spanned the length of their 3'-regions were examined. In SERCA2a, this fragment respresents the last 108 bases of the mRNA sequence (2A6). In SERCA2b, serveral unstable regions were indentified but the most unstable fragment is within the first 300 bases after the splice site (2B1). Experiments using hybrid fragments with 2A6 and 2B1, however, showed that the effects of these sequences on RNA decay may be context dependent. The cis-acting elements function by binding to trans-acting factors (RNA-binding proteins). The ability of the large overlapping 3'-region fragments from SERCA2a and 2b to bind proteins was determined using electrophoretic mobility shift assays. Only one fragment within SERCA2a did not bind to at least one protein while all the SERCA2b fragments were able to bind proteins. Using small RNA oligonucleotides to compete for protein binding with 2A6, we showed that hairpin loops in this region may be involved in protein binding. In contrast, the GC-rich coding region sequence from 3521-3555 appears to contain the key cis-acting element in SERCA2b (2B1). As several fragments downstream of 2B1 were also unstable, this cis-acting element may act in conjunction with other downstream sequences. In summary, the 3'-region plays a role in the predominantly nuclear decay of SERCA2a and 2b in LVM and SSM via specific cis-acting elements and trans-acting factors. This decay may be important in development and disease. Doctor of Philosophy (PhD) |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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