Date of Award

2011

Degree Type

Thesis

Department

Biological, Geological and Environmental Sciences

First Advisor

Shukla, Girish

Subject Headings

Oncogenes, RNA splicing, Messenger RNA, Nucleoproteins, Breast -- Cancer, HER-2 gene, Splicing, RNA, breast carcinoma, HER-2

Abstract

In nuclear pre-mRNA splicing, introns are removed through cooperative interactions of small nuclear RNAs (snRNAs). Many human genes are interrupted by two types of nuclear pre-mRNA introns. Major class or the U2-dependent type introns are spliced by U1, U2, U4, U5 and U6 snRNAs. The minor class or U12-dependent type introns are spliced by U11, U12, U4atac, U5, and U6atac snRNAs. It has been shown that over expression of minor class spliceosomal snRNAs can have an inhibitory effect on the splicing of major class introns. To further test the concept, we targeted HER-2/Neu proto-oncogene, which is over-expressed in 20-30 of human mammary tumors. We constructed a series of mutant human U6atac and U11 snRNAs to target 5' splice site of introns 1, 2, 6, 8, 12 and 13 of HER-2/Neu in order to prevent nuclear pre-mRNA splicing and to down regulate protein synthesis. To determine the efficacy of our approach, we transiently expressed mutant snRNAs in mammary carcinoma cells to activate splicing interference (SPLICEi) of HER-2/Neu pre-mRNA. Our data indicates that the modulation of HER-2/Neu pre-mRNA by mutant snRNAs mediated SPLICEi affect the protein expression in cultured cells. Furthermore, albeit modest, we observed inhibition of cancer cell proliferation and activation of apoptosis. Although, our approach is still at experimental stages, it presents another opportunity for targeting of oncogenes at RNA level

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