An Exonic Splicing Silencer Is Involved in the Regulated Splicing of Glucose 6-Phosphate Dehydrogenase mRNA

Abstract

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.Glucose-6-phosphate dehydrogenase (G6PD) 2 is a member of a family of enzymes that catalyze the de novo synthesis of fatty acids. In liver, the lipogenic pathway plays an essential role in converting excess dietary energy into a storage form. Consistent with this role in energy homeostasis, the capacity of this pathway is regulated by dietary changes, such as fasting, feeding, and the amount and type of carbohydrate and polyunsaturated fat in the diet (1). For many of the lipogenic enzymes, regulation of enzyme amount occurs primarily by changes in the transcription rate of the gene, but posttranscriptional regulation via mRNA stability has also been implicated (1). G6PD differs from the other family members in that dietary regulation occurs exclusively at a posttranscriptional step (2-4).G6PD expression is inhibited by polyunsaturated fatty acids, such as arachidonic acid; this occurs at a unique posttranscriptional step involving a decrease in the rate of splicing of the nascent G6PD transcript. Several lines of evidence indicate that changes in mature mRNA accumulation are caused by changes in the efficiency of splicing of the G6PD transcript. First, changes in the cytoplasmic accumulation of G6PD mRNA are preceded by changes in the accumulation of mRNA in the nucleus in the absence of changes in transcriptional activity of the gene (3-5). Second, stimulatory treatments, such as refeeding, enhance the ...

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

“…The G6PD regulatory sequence does score highly as a potential exonic regulatory sequence using a new algorithm developed using computational analysis of 46,103 exons [45]. This is consistent with our most recent data demonstrating that the region from nt 43-72 of exon 12 is an ESS [46].…”

“…Thus, these proteins either do not bind within this sequence or the amount bound was below the limits of detection. Because hnRNPs L and A2/B1 are known to bind ESSs and act as splicing silencers, the binding of these proteins to G6PD pre-mRNA is consistent withthis element being an ESS [46].…”

Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver

“…The increase in G6PD mRNA results in greater expression of the enzyme and an increase in lipogenic capacity as compared with fasted animals (13). These in vivo feeding experiments are recapitulated in primary rat hepatocytes in culture in which treatment with insulin induces the accumulation of spliced G6PD mRNA, and the polyunsaturated fatty acid, arachidonic acid, attenuates mRNA splicing (2,15,16). A splicing regulatory element in exon 12 of the G6PD transcript mediates the effect of nutrients upon the splicing of this precursor mRNA (2).…”

“…Although key enzymes in the pathways of intermediary metabolism are primary targets for regulation by nutritional status, our current understanding of the molecular details causing this regulation have emphasized transcriptional mechanisms or changes in the kinetic properties of key regulatory enzymes. We have gathered a body of data indicating that alternative RNA splicing is a significant, yet underappreciated mechanism by which nutrients regulate gene expression (1,2).…”

“…These in vivo feeding experiments are recapitulated in primary rat hepatocytes in culture in which treatment with insulin induces the accumulation of spliced G6PD mRNA, and the polyunsaturated fatty acid, arachidonic acid, attenuates mRNA splicing (2,15,16). A splicing regulatory element in exon 12 of the G6PD transcript mediates the effect of nutrients upon the splicing of this precursor mRNA (2). The absence of transcriptional regulation of this gene by nutrients has made it a useful model for deciphering the molecular mechanisms involved in this post-transcriptional regulation.…”