Colleen Kirby
Illinois Wesleyan University
Bloomington, IL
Faculty Mentor: Dr. Stephen Munroe

mRNA splicing is a critical step in the expression of genes in eukaryotic organisms. The primary RNA transcript is a faithful copy of the gene, containing both exons (sequences retained in mRNA) and introns (nucleotides not retained). During splicing, the introns are cut out and the exons are joined together to produce an mRNA molecule that codes directly for a protein. Alternative processing is a possible outcome from splicing and results in variant forms of mature mRNAs. Alternative processing requires the recognition and selection of suboptimal splice and is facilitated by splicing enhancer sequences.

Alternative processing of thyroid hormone receptor (TR) mRNA results in two antagonistic protein products. One product, TRa1, binds to the thyroid hormone while the other protein, TRa2, lacks the functional binding site and does not mediate a hormonal response. TRa2 mRNA arises from splicing out most of the upstream TRa1 specific 3’ terminal exon and including a TRa2 specific alternative exon. The alternative processing of TR mRNA is regulated by multiple splicing enhancers. One exonic splicing enhancer, ESX10, is located in a specific region of TRa2 mRNA that overlaps with another mRNA encoded by the RevErbAa gene. These genes are unusual because both strands of the overlap code for proteins. The goal of this research is to understand the sequence requirements for ESX10 enhancer activity and study its evolution. By determining whether or not a sequence homologous to the RevErbA gene includes the sequence for ESX10, it can be ascertained whether the mammalian TRa2 mRNA evolved in response to the preexisting enhancer or if the splice site evolved first and the enhancer evolved after TRa2 splicing was established.

One way to examine the evolutionary development of ESX10 in TRa2 is to compare the corresponding antisense sequence of RevErbAa with that of the homologous gene RevErbAa. The RevErbAa and b loci evolved as a consequence of an early gene duplication and retain 60% similarity. One important difference is that while RevErbAa mRNA overlaps the TRa2 transcript, there is no overlap between TRb and RevErbAa. By testing RevErbAb sequences in a heterologous pre-mRNA construct, it can be determined if the RevErbAb antisense sequences have the enhancer activity of ESX10 antisense to RevErbAa. If RevErbAb sequence has less splicing enhancer activity than the corresponding RevErbAa sequence, then the splicing enhancer probably evolved in TRa in response to the preexisting alternative splice site. However, if the RevErbAb sequence has splicing activity comparable to RevErbAa, then ESX10 was probably associated with the ancestral gene prior to evolution of TRa2. Experiments to test these two models will be described.