Poster abstracts

Poster number 85 submitted by Callee M. Walsh

Juxtaposed ESE and ESS elements in a constitutive exon regulate mRNA splicing.

Callee M. Walsh (West Virginia University), Wioletta Szeszel-Fedorowicz (West Virginia University), Alison B. Kohan (West Virginia University), Lisa M. Salati (West Virginia University)

Abstract:
Alternative splicing of eukaryotic mRNAs increases the number of proteins that are produced from a single gene. Dietary status and individual nutrients are potent regulators of alternative splicing as we observe with glucose-6-phosphate dehydrogenase (G6PD). G6PD expression is regulated by intron retention in which the rate of intron removal and splicing determines the amount of the translated protein. The amount of G6PD mRNA changes in response to nutrient stimuli such that insulin and dietary carbohydrate enhance intron removal, which increases the accumulation of mature mRNA. Starvation and polyunsaturated fatty acids decrease the rate of intron removal, leading to intron retention. These properties make G6PD an excellent model to determine how nutrients regulate pre-mRNA splicing. Exon 12, a constitutive exon of G6PD mRNA, contains an ESS within nucleotides 43-72 that inhibits intron removal. Through RNA affinity chromatography and proteomic approaches, hnRNPs K, L, and A2/B1 were identified as putative regulatory proteins. We hypothesize that these hnRNPs are splicing silencer proteins, as we have observed greater binding of the hnRNPs to the regulatory element during starvation, corresponding to a reduction in splicing, than during re-feeding when splicing is enhanced. The 30 nucleotide regulatory element in exon 12 also contains an ESE. Deletion of the entire element from an in vitro splicing substrate does not restore splicing. Consistent with the regulatory element containing an ESE, binding of SRp20 and SRp75 was detected by RNA affinity chromatography; furthermore, binding of SRp20 is enhanced by re-feeding, corresponding to an increase in splicing. In hepatocytes, insulin stimulates phosphorylation of several SR proteins while addition of arachidonic acid inhibits this effect. Thus, nutrients can regulate the activity of splicing regulatory proteins causing changes in the splicing of G6PD and potentially other pre-mRNAs. We hypothesize that mutually exclusive binding of hnRNPs and SR proteins regulates this model of intron retention. [Supported by DK046897 and a WV Graduate Fellowship in Science, Technology, Engineering and Math.]

Keywords: splicing, nutrients, SR proteins