The molecular mechanism of glucose-6-phosphate dehydrogenase regulation by dietary factors in intact animals.

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 matrixâ€. 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

Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex

Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3′ splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex

Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metab., including the repression of alternative exons. We have detd. the soln. structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two sepd. pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor. [on SciFinder (R)