Splicing Signals Are Required for S-Phase Regulation of the Mouse Thymidylate Synthase Gene

Abstract

The thymidylate synthase (TS) gene is expressed at a much higher level in cells undergoing DNA replication than in nondividing cells. In growth-stimulated mammalian cells, TS mRNA content increases 10 to 20-fold as cells progress from G 1 through S phase. However, the rate of transcription of the TS gene does not increase during this interval, indicating that the gene is regulated at the posttranscriptional level. We have shown that both the promoter of the mouse TS gene and TS introns are necessary (although neither is sufficient) for S-phase-specific regulation of TS mRNA content. In the present study, we examined in more detail the role of introns in regulating TS mRNA levels in growth-stimulated cells. TS minigenes that contain normal or modified introns were stably transfected into mouse 3T6 fibroblasts, and the regulation of the minigenes was compared with that of the endogenous TS gene. TS minigenes that contain TS intron 1 or 2 maintain S-phase regulation. Deletion of most of the interior of the introns had only minor effects on regulation. However, when splicing of the intron was inhibited by alteration of the splice donor and acceptor sites, the minigene was expressed at a constant level following growth stimulation. Minigenes consisting of the TS promoter linked to either a luciferase or a human ␤-globin indicator gene were growth regulated when spliceable introns were included in the minigenes. However, when the introns were eliminated, the minigenes were expressed at a constant level. These observations indicate that the splicing reaction itself, rather than a control sequence within the intron, is important for growth-regulated expression of the TS gene. Possible mechanisms to account for the dual requirement for the TS promoter and intron splicing for proper regulation of the TS gene are discussed.During the past few years, considerable progress has been made in determining the mechanisms that control the expression of S-phase genes, which encode enzymes that are important for DNA synthesis. Such genes are expressed at a high level when cells are undergoing DNA replication but at a much lower level in quiescent cells or during G 1 phase of the cell cycle (13). Detailed analyses have been performed on several S-phase genes, including those that encode thymidine kinase, dihydrofolate reductase, proliferating cell nuclear antigen, and DNA polymerase ␣. Nuclear run-on assays and analyses of the expression of indicator genes driven by promoters of these genes have shown that the rate of transcription of many Sphase genes increases near the G 1 -S-phase boundary (e.g., 7, 32, 45-48). Members of the E2F family of transcription factors appear to play an important role in coordinating the transcription of many S-phase genes with entry into S phase (16,35,42). In addition, many S-phase genes are also controlled by posttranscriptional mechanisms. Regulation has been observed at the level of RNA processing, mRNA stability, mRNA translation, and enzyme stability (e.g., 9, 12, 23, 31, 44).We have been analyzing...

Inhibition of the Splicing of Glucose-6-phosphate Dehydrogenase Precursor mRNA by Polyunsaturated Fatty Acids

“…These include thymidylate synthase (41), tumor necrosis factor-␣ (42), and spot 14 (43,44). Regulated expression of thymidylate synthase during the cell cycle requires both the thymidylate synthase promoter and a spliceable intron, which need not be from the thymidylate synthase RNA (41). In contrast, splicing of the tumor necrosis factor-␣ mRNA requires a cis-acting element in the 3Ј-UTR of the transcript (42).…”

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

“…In contrast to the situation with many other mammalian S phase genes, TS gene expression is controlled primarily at the post-transcriptional level, rather than the transcriptional level [Johnson, 1994;Ayusawa et al, 1986]. Analyses of stably transfected TS minigenes have shown that sequences in the promoter region as well as an intron in the transcribed region are both necessary (but neither is sufficient) for proper S phase accumulation of TS mRNA Ash et al, 1993Ash et al, , 1995Ke et al, 1996]. This finding suggests that some form of communication between the promoter and RNA processing machinery is necessary for proper regulation of TS mRNA production during the G1 to S phase transition.…”

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

“…This process is highly regulated. An increasing number of genes have been identified whose expression is regulated by changes in the rate of splicing of constitutive exons; G6PD is one such gene [2,[37][38][39]. Unique to the regulated splicing of G6PD is that it occurs in response to nutritional cues [2].…”