E2F-dependent mitogenic stimulation of the splicing of transcripts from an S phase-regulated gene

Abstract

There is one class of genes whose expression increases at the G1/S transition of the cell cycle. One of these genes codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), an enzyme that controls glycolysis. The cell-cycle regulation of the PFK-2 gene depends on a binding site for the transcription factor E2F located at the 5' end of the first exon and involves not only a transcriptional, but also a post-transcriptional, mechanism. We have investigated this mechanism by studying in Rat-1 fibroblasts mature and immature mRNAs from the endogenous PFK-2 gene and from stably expressed transgenes containing PFK-2 gene regions. An increase in precursor mRNA half-life and processing took place at the G1/S transition. Transgenes with a mutated E2F binding site or with mutated splice sites lost the regulation by serum, indicating that both an intact E2F binding site and an efficient splicing reaction are necessary for proper mitogenic stimulation. In quiescent cells, the transgene lacking the E2F binding site was more efficiently spliced than the wild-type construct. These results indicate that, in the wild-type gene, precursor mRNA splicing is blocked in G0 and that this block requires the E2F binding site. The data provide evidence for a coupling between stimulation of promoter activity and increased mRNA splicing in the mitogenic regulation of S phase-regulated genes.