Characterization of regulatory mechanisms for alternative splicing in alpha thyroid hormone receptor mRNA

In eukaryotes, alternative splicing is an essential post transcriptional modification process for functional gene expression and a major contributor to protein diversity. The regulation of alternative splicing generally involves the engagements of RNA sequences cis-acting elements) and corresponding protein factors (trans-acting factors). The cis-acting RNA motifs can be categorized depending on positional and functional differences. Trans-acting protein factors will then bind to RNA sequences and affect the corresponding splicing activity. Recently, factors associated with 3' polyadenylation have also been identified to affect alternative splicing.;In mammals, the alpha-thyroid hormone receptor gene (TRalpha) produces transcripts for two functionally antagonistic isoforms, TRalpha1 and TRalpha2 by alternative splicing of pre-mRNA. TRalpha1 will activate the thyroid hormone responsive genes after the binding of thyroid hormone while TRalpha2, a non-hormone binding variant, plays a functionally antagonistic role. A third minor isoform, TRalpha3 has also been described which is similar to TRalpha2 but lacks part of the terminal sequence. Regulation of TRalpha alternative splicing requires the interaction of cis-trans elements and alternative polyadenylation. The goal of my project is to study the regulatory mechanism of TRalpha alternative splicing.;In our system, ESX10 (exonic splicing enhancer on exon10) was previously identified as a 200 nt splicing enhancer element located on the last exon of TRalpha gene. My study further characterized the enhancing capability of exonic splicing enhancers (ESE) motifs within ESX10. Three heptanucleotide ESE motifs in the 3' half of ESX10 have been identified. In vitro experiments indicate that substitution of eight nucleotides within three heptamers decreases the enhancing capability of ESX10. After substitution of ESX10 and its subfragments with Rev-erbbeta sequence, cryptic splicing was detected and quantitated. Previous experiments also indicate that replacing original TRalpha1 and TRalpha2 poly (A) site with a strong SVL poly (A) signal would increase the corresponding mRNA expression. My results show that introduction of downstream poly (A) signal increases splicing fidelity. When upstream 5'ss of TRalpha2 is disabled, the strong poly (A) signal enhances the usage of weak splicing cites and promote cryptic splicing.