Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

Gudas, Jean M.; Knight, Glenn B.; Pardee, Arthur B.

doi:10.1073/pnas.85.13.4705

Cited by 76 publications

(50 citation statements)

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“…For example, the splicing of TS transcripts may be much more efficient in S phase than in GdG1 phase. This possibility is in line with the conclusions of Gudas and coworkers (12,13), who studied the processing of thymidine kinase (TK) mRNA in growthstimulated cells. They showed that TK mRNA as well as various nuclear splicing intermediates increase to a much greater extent than does the rate of transcription of the gene as cells progress from Go to S phase.…”

Section: Discussionsupporting

confidence: 76%

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

Ash

Korb

et al. 1993

Mol. Cell. Biol.

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The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human 1-globin gene, and the bovine growth hormone gene were stable transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G, through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.When quiescent mammalian cells are stimulated to reenter the cell cycle following exposure to a mitogen, changes in the pattern of gene expression occur which lead to the initiation of DNA replication and cell division. Early changes include the activation of genes such asfos and myc, which encode transcriptional regulatory proteins. Later in G1, after the cells have become committed to enter S phase, genes that are necessary for DNA replication are activated (7). There is considerable interest in understanding the mechanisms by which the S-phase genes are controlled and how these controls are related to the signals that govern G, progression (20).We have been studying the mechanisms for regulating the expression of the S-phase gene that encodes thymidylate synthase (TS). TS is an essential housekeeping enzyme that catalyzes the reductive methylation of deoxyuridylic acid to form thymidylic acid in the de novo biosynthetic pathway. The mouse TS gene is 12 kb in length and has a 1-kb coding region that is interrupted by six introns (4, 30). The G/C-rich promoter region lacks a TATAA box and initiates transcription at multiple sites over a 60-nucleotide region (4, 8). Mouse TS mRNA is highly unusual in that the predominant species lacks a 3' untranslated region (18). The poly(A) tail of the mRNA extends directly from the UAA termination codon. The upstream polyadenylation signal is ATTAAA and is located within the coding...

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Section: Discussionsupporting

confidence: 76%

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

Ash

Korb

et al. 1993

Mol. Cell. Biol.

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“…Introns 1 and 4 of the proliferating cell nuclear antigen gene appear to be important for proper down-regulation of the gene (1,44). Alterations in splicing mechanisms also appear to be important for controlling thymidine kinase gene expression (23,24).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Ash

Johnson

1996

Molecular and Cellular Biology

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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...

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“…Total RNA was prepared by lysing cell monolayers in guanidinium isothiocyanate and centrifuging them over a 5.7 M CsCl cushion as described previously (8). RNA (20 g) was electrophoresed on denaturing formaldehyde gels, transferred to MagnaNT membranes and crosslinked with UV.…”

Section: Methodsmentioning

confidence: 99%

Cyclin E2, a Novel G₁ Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

Gudas

Payton

Thukral

et al. 1999

Molecular and Cellular Biology

165

147

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A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27Kip1 and p21 Cip1 . The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G 1 /S transition. Overexpression of cyclin E2 in mammalian cells accelerates G 1 , demonstrating that cyclin E2 may be rate limiting for G 1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.The eukaryotic cell cycle is regulated by a family of cyclindependent kinases (CDKs) that are cyclically activated to trigger the different phases of the cell cycle in the correct order and at the right time. CDKs are regulated by a number of different proteins, including the cyclins that bind and activate the CDKs to form a serine/threonine kinase holoenzyme complex. In mammals, D-type cyclins in conjunction with cyclins E and A are required for cells to traverse G 1 and enter S phase (19,25,29). There are three members of the D-type cyclins, two members of the cyclin A family, and, to date, one mammalian cyclin E gene (33). The formation of distinct G 1 cyclin-CDK complexes regulates the temporal phosphorylation of specific substrates that drive cells through G 1 and into S phase. Cyclin-CDK complexes are negatively regulated by two families of CDK inhibitors (34). The Ink4 family of CDK inhibitors exclusively regulate D-type cyclin-CDK complexes, while the Kip/Cip family regulate D-type cyclins as well as CDK complexes comprised of cyclin E or A. p27Kip1 and p21 Cip1 bind and inhibit cyclin E-Cdk2 complexes by acting as competitive inhibitors for substrates and by preventing cyclin-activating kinase (CAK)-mediated phosphorylation and activation of Cdk2 (23,34).Cyclin E was originally discovered by its ability to complement the G 1 cyclins in budding yeast (16,18). This observation suggested that cyclin E regulated the progression of cells through the cell cycle. It was later shown that cyclin E-Cdk2 complexes stimulate mammalian cells to traverse G 1 and enter S phase by the temporal phosphorylation of specific substrates such as the retinoblastoma tumor suppressor protein (Rb) (17,20,33). In fission yeast, a critical size must be reached prior to entry into mitosis, and this is normally regulated by coupling Cdk1 activation to cell growth. Premature activation of Cdk1 in fission yeast causes cells to enter mitosis at a reduced size. A similar effect occurs when cyclin E is overexpressed three-to fourfold in mammalian cells; the cells have a shorter G 1 and enter mitosis at a reduced size (25, 29). These resul...

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Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

Cited by 76 publications

References 50 publications

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

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

Cyclin E2, a Novel G₁ Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

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Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

Cited by 76 publications

References 50 publications

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

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

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

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Cyclin E2, a Novel G₁ Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers