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The expression of glucose-6-phosphate dehydrogenase (G6PD) gene is stimulated by a high-carbohydrate diet and it is inhibited by the addition of polyunsaturated fat to the high-carbohydrate diet. G6PD expression is regulated by a posttranscriptional mechanism in the nucleus. In this regard, changes in the amount of G6PD mRNA in the cytoplasm are preceded by similar changes in the amount of nuclear precursor mRNA (pre-mRNA) in the absence of changes in transcriptional activity of the gene. Some components of the transcription and pre-mRNA processing machinery as well as pre-mRNA itself are associated with the insoluble portion of the nucleus known as “nuclear matrix”. To determine whether the processing of G6PD pre-mRNA is regulated by dietary factors, the nuclear matrix RNA was isolated. Using probes that cross intron-exon boundaries, we measured G6PD mRNA species with the intron (less-processed) or from which the intron was spliced (more-processed). Feeding the high-carbohydrate diet increased the abundance of G6PD pre-mRNA on the matrix and resulted in a larger increase in both the abundance of more-processed mRNA and its rate of accumulation. Further, using probes that cross two consecutive exon-intron boundaries, we observed that partially spliced mRNA was increased to a greater extent and at a faster rate than its precursor. These results are consistent with a stabilization of G6PD mRNA during the splicing. In contrast, with spot 14, a gene regulated primarily by transcriptional changes, pre- and mature mRNA increased in parallel during the refeeding. Addition of the polyunsaturated fat to the diet decreased the abundance of G6PD mature mRNA 50% in the cytoplasm and in the nuclear matrix fraction. The abundance of G6PD premRNA was similar during the first 2 h of the daily eating cycle in mice fed either the low-fat or the high-fat diet. Yet, the amount of G6PD more-processed RNA was inhibited in mice consuming the high-fat diet. The abundance of pre-mRNA increased after 4 h of the low-fat diet and this increase was attenuated by the high-fat diet. The results suggest that, polyunsaturated fatty acids decrease both the processing and the accumulation of the G6PD pre-mRNA most likely by destabilizing the pre-mRNA.