Date of Award

Spring 2003

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Pre-mRNA transcripts of the thyroid hormone receptor gene, erbAa, are alternatively processed to generate two different protein products: TRal and TRa2. One of the proteins, TRal, is the functional receptor that binds to thyroid hormone (T3) and to thyroid response elements (TREs) associated with target genes. TRa2 is the variant receptor protein that lacks the T3 binding site but is able to bind TREs. This protein does not elicit a hormonal response and therefore is used to down regulate the T3 signal. Alternative processing occurs by use of different splice patterns and by use of different polyadenylation (poly A) sites. There is a suboptimal 5' splice site (ss) within I exon 9 of the erbAa gene that is utilized when forming TRa2. When this occurs, exon 10 is polyadenylated and a variant mRNA is formed. However, if splicing does not occur within exon 9, ex on 9 becomes polyadenylated and TRa1 mRNA is generated. There are " different RNA sequence elements known as splicing enhancers that regulate the use of the splice sites. These sequences are typically purine rich and bind various proteins that control splice site utilization. The goal of my project was to investigate regulation of alternative processing in the erbAa gene which included the identification and characterization of splicing enhancers. Competition between polyadenylation at the end of exon 9 and use of an upstream suboptimals 5'ss was observed by mutating the polyadenylation site. :The competition that occurs between the two sites is crucial for regulating the production of each mRNA. T also identified sequences that are necessary for maximal splicing t enhancenient by an intronic splicing enhancer, TR-ISE3, that is located near the 3' ss of exon 10. This was accomplished by making deletions within the enhancer region of intron 9. Lastly, by making a deletion within exon 10 I identified a strong exonic splicing enhancer. This enhancer, ESXIO, is located within the overlap region between the erbAa and RevErbAa genes and was shown to enhance splicing both in vivo and in vitro.

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