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J. Biol. Chem., Vol. 281, Issue 45, 34146-34158, November 10, 2006

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

From the Department of Biochemistry and Molecular Pharmacology, West Virginia University, Morgantown, West Virginia 26506

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.

Received for publication, April 20, 2006 , and in revised form, September 14, 2006.

^* This work was supported by National Institutes of Health Grant DK46897 (to L. M. S.), American Heart Association Grant 0315129B (to B. N. G.), the COBRE for Signal Transduction and Cancer (National Institutes of Health Grant P20RR016440), and the West Virginia University Proteomics Facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Pharmacology, West Virginia University Health Sciences Center, P.O. Box 9142, Morgantown, WV 26506. Tel.: 304-293-7759; E-mail: Lsalati{at}hsc.wvu.edu.

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