Trisha Haubrich
Marquette University
Milwaukee, WI
Faculty Mentor: Dr. Stephen Munroe

The thyroid hormone receptor gene, erbAa, in mammals encodes two isoforms that arise from alternative splicing. These two receptors, TRa1 and TRa2, are functionally antagonistic. TRa1 possesses a hormone binding domain, whereas TRa2 does not. TRa1 binds thyroid hormone, T3, at its hormone binding domain, which activates transcription in the cell. TRa2 does not have a hormone binding domain and is therefore unresponsive to T3.

TRa2 is formed during alternative splicing when a portion of the gene including the 3' end of TRa1 is removed by splicing. The TRa2-specific 5' splice site is located in exon 9 of erbAa. TRa2 also includes exon 10, which is not a part of TRa1. Previously, a splicing enhancer, designated SEa2, was found downstream of the TRa2 5' splice site. Through deletion experiments, an 80-nucleotide portion (SE 80) of the enhancer was determined to be necessary for TRa2 splicing. Activity of the splicing enhancer changed little when 20 nucleotides were deleted from this region (SE 60), however it made the mutation convenient to work with. SE 60 contains a splice site-like sequence, and a single base mutation (+6 U) makes the sequence resemble the 5' consensus splice site and has been found to activate cryptic splicing.

To study the role of the pseudo 5' splice site in the activity of the splicing enhancer regulating the erbAa gene's alternative splicing and therefore controlling the balance between TRa1 and TRa2 in the cell, several different plasmids were constructed. The plasmids were made with different combinations of the SE 60, the +6 U point mutation, and a mutation that inactivates the TRa2-specfic 5' splice site. These plasmids were transfected into 293 cells. Transfections were also carried out in parallel with several previously characterized control constructs. The RNA was isolated from the cells and analyzed by RNase protection assays using specific plasmid probes to monitor the levels of splicing and cryptic use of the pseudo 5' splice site with the SEa2 splicing enhancer. The results of these experiments will provide information on possible interactions between the 5' splice site specific for TRa2 mRNA and the pseudo 5' splice site associated with the splicing enhancer.