Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD-kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.

SUMMARY Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD. Received July 27, 1998; revised version accepted September 28, 1998. Eukaryotic pre-mRNAs are transcribed by RNA polymerase II (Pol II) and undergo several processing events before becoming mature mRNA. These events, including 5Ј capping, splicing, and polyadenylation, are completed in the nucleus before the mRNA is transported to the cytoplasm and translated. Capping of the 5Ј end of the mRNA is the first detectable mRNA processing event, occurring by the time the transcript is only 25-30 nucleotides long (Jove and Manley 1984;Rasmussen and Lis 1993). This cotranscriptional event is mediated by recruitment of the capping enzyme machinery to the phosphorylated carboxy-terminal domain (CTD) of the largest subunit of Pol II McCracken et al. 1997;Yue et al. 1997;Ho et al. 1998).Capping occurs by a series of three enzymatic reactions. The 5Ј triphosphate end of the nascent RNA Pol II transcript is cleaved by 5Ј RNA triphosphatase to produce a diphosphate terminus. RNA guanylyltransferase forms a covalent enzyme-GMP complex and subsequently caps the RNA substrate by adding the guanosine residue in a 5Ј-5Ј triphosphate linkage. The cap is then methylated at the guanine N7 position by RNA (guanine-7) methyltransferase, completing the m 7 GpppN, or cap0, structure (for review, see Mizumoto and Kaziro 1987;Shuman 1995). In higher eukaryotes, a bifunctional monomeric polypeptide carries both RNA triphosphatase and guanylyltransferase activities . Recent characterizations of the capping enzymes from Caenorhabidits elegans, mouse, and human reveal an amino-terminal RNA triphosphatase domain and a carboxy-terminal guanylyltransferase domain (McCracken et al. 1997;Takagi et al. 1997;Wang et al. 1997;Yue et al. 1997;Tsukamoto et al. 1998;YamadaOkabe et al. 1998). In contrast, the yeast Saccharomyces cerevisiae capping enzyme is a heterodimer. The CET1 gene encodes the 62-kD triphosphatase subunit (Tsukamoto et al. 1997), and the CEG1 gene encodes the 52-kD guanylyltransferase subunit (Itoh et al. 1987;Shibagaki et al. 1992). Both CET1 and CEG1 genes are essential for viability.The guanylyltransferase mechanism is conserved among eukaryotes and virus, involving a covalent enzyme-guanylate intermediate in which GMP is linked to the ⑀-amino group of the active site lysine (for review, see Mizumoto and Kaziro 1987). The recently solved structures of the Chlorella virus PBCV-1 guanylyltransfe...

mRNA capping requires the sequential action of three enzymatic activities: RNA triphosphatase, guanylyl-transferase, and methyltransferase. Here we characterize a gene (CEL-1) believed to encode the C. elegans capping enzyme. CEL-1 has a C-terminal domain containing motifs found in yeast and vaccinia virus capping enzyme guanylyltransferases. The N-terminal domain of CEL-1 has RNA triphosphatase activity. Surprisingly, this domain does not resemble the vaccinia virus capping enzyme but does have significant sequence similarity to the protein tyrosine phosphatase (PTP) enzyme family. However, CEL-1 has no detectable PTP activity. The mechanism of the RNA triphosphatase is similar to that of PTPs: the active site contains a conserved nucleophilic cysteine required for activity. These results broaden the superfamily of PTP-like phosphatases to include enzymes with RNA substrates.

In the course of studying the ST2 gene, which was initially found to be expressed specifically at the C&G, transitional state in BALBlc-3T3 cells and was one of the primary response genes, we found another ST2-related mRNA, designated as STZL, in serum-stimulated BALB/c-3T3 cells in the presence of cycloheximide. Nucleotide sequence analysis of the cloned STZL cDNA revealed that it had an open reading frame encoding a polypeptide of 567 amino acids. A 5' region (1,028 nucleotides) of STZL cDNA was identical with the ST2 cDNA, and a unique 3' region encoded 'a putative transmembrane domain of 24 amino acids and a cytoplasmic domain of 201 amino acids. The ST2 gene product is highly similar to the extracellular portion of IL-I receptors type 1 and type 2, and the STZL gene product shows a marked similarity with entire IL-l receptor type I.

Dietary sugars are known to stimulate intestinal glucose transport activity, but the specific signals involved are unknown. The Na(+)-dependent glucose co-transporter (SGLT1), the liver-type facilitative glucose transporter (GLUT2) and the intestinal-type facilitative glucose transporter (GLUT5) are all expressed in rat jejunum [Miyamoto, Hase, Taketani, Minami, Oka, Nakabou and Hagihira (1991) Biochem. Biophys. Res. Commun. 181, 1110-1117]. In the present study we have investigated the effects of dietary sugars on these glucose transporter genes. A high-glucose diet stimulated glucose transport activity and increased the levels of SGLT1 and GLUT2 mRNAs in rat jejunum. 3-O-Methylglucose, D-galactose, D-fructose, D-mannose and D-xylose can mimic the regulatory effect of glucose on the SGLT1 mRNA level in rat jejunum. However, only D-galactose and D-fructose increased the levels of GLUT2 mRNA. The GLUT5 mRNA level was increased significantly only by D-fructose. Our results suggest that the increase in intestinal transport activity in rats caused by dietary glucose is due to an increase in the levels of SGLT1 and GLUT2 mRNAs, and that these increases in mRNA may be caused by an enhancement of the transcriptional rate. Furthermore, for expression of the SGLT1 gene, the signal need not be a metabolizable or transportable substrate whereas, for expression of the GLUT2 gene, metabolism of the substrate in the liver may be necessary for signalling. Only D-fructose is an effective signal for expression of the GLUT5 gene.

A human cDNA was isolated encoding a protein with significant sequence similarity (41% identity) to the BVP RNA 5-phosphatase from the Autographa californica nuclear polyhedrosis virus. This protein is a member of the protein-tyrosine phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998) J. Biol. Chem. 272, 20347-20353) to be a nuclear protein that can associate with RNA or ribonucleoprotein complexes. We demonstrate that PIR1 removes two phosphates from the 5-triphosphate end of RNA, but not from mononucleotide triphosphates. The specific activity of PIR1 with RNA is several orders of magnitude greater than that with the best protein substrates examined, suggesting that RNA is its physiological substrate. A 120-amino acid segment Cterminal to the PTP domain is not required for RNA phosphatase activity. We propose that PIR1 and its closest homologs, which include the metazoan mRNA capping enzymes, constitute a subgroup of the PTP family that use RNA as a substrate.The protein-tyrosine phosphatase (PTP) 1 superfamily includes a large number of enzymes that dephosphorylate diverse substrates including proteins, nucleic acids, and lipids (1-6). Members of the PTP superfamily are thought to use a common catalytic mechanism involving the formation and subsequent hydrolysis of a phosphocysteine intermediate (1-6). The essential Cys and Arg residues are located within an active site motif (HCX 5 R) that characterizes all phosphatases of this superfamily.The Autographa californica nuclear polyhedrosis virus expresses a 19-kDa phosphatase of the PTP superfamily designated herein as BVP (also known as BVH1 and BVPTP) (7-9). BVP was originally characterized as a dual specificity protein phosphatase (7-9), but subsequent studies have demonstrated that its RNA phosphatase activity is several orders of magnitude greater than its activity with protein substrates (3, 4). BVP shares significant sequence similarity with the RNA triphosphatase domain of the metazoan mRNA capping enzymes (2-4, 10). The bifunctional capping enzymes of metazoa contain an N-terminal RNA 5Ј-triphosphatase domain and a C-terminal GTP::RNA guanylyltransferase domain. The RNA triphosphatase domain removes the ␥-phosphate from the 5Ј end of nascent mRNA to leave a diphosphate terminus and the guanylyltransferase domain catalyzes the subsequent transfer of a guanylyl group from GTP to produce the unmethylated 5Ј cap structure, G(5Ј)ppp(5Ј) N (11, 12). BVP and the triphosphatase domains of the capping enzymes contain the signature HCX 5 R active site motif common to all PTPs and are thought to employ a catalytic mechanism similar to that used by PTPs to dephosphorylate proteins (2-4, 10, 13, 14). BVP differs from the metazoan capping enzymes in that it lacks a guanylyltransferase domain and releases both ␥-and ␤-phosphates from mRNA to yield a monophosphate at the 5Ј end (3). BVP is unlikely to be involved in the capping of viral messages because LEF-4, a subunit of the A. californica nuclear ...

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5-phosphatase. BVP sequentially removes ␥ and ␤ phosphates from the 5 end of triphosphate-terminated RNA, leaving a 5-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.Recently, several mRNA capping enzyme genes from higher eukaryotes have been described (refs. 1-6; reviewed in ref. 7). These proteins consist of two domains, each with distinct enzymatic activity. The C-terminal guanylyltransferase domain contains motifs found in yeast and viral mRNA guanylyltransferases as well as DNA and RNA ligases (reviewed in refs. 8 and 9). Surprisingly, the N-terminal domain exhibits significant sequence similarity to the protein tyrosine phosphatase (PTP) family of enzymes but does not have appreciable PTP activity. However, it does have 5Ј-RNA triphosphatase activity that produces the RNA 5Ј-diphosphate necessary for subsequent guanylylation (1,3,5,6,10).The PTPs are a large family of enzymes that catalyze the hydrolysis of phosphotyrosine from various proteins. They have been implicated as key players in signaling pathways controlling metabolism, cell growth, differentiation, and cytoskeletal dynamics (reviewed in refs. 11-13). All members of this family have the highly conserved HCX 5 R active site motif. The invariant cysteine residue within this consensus sequence acts as a nucleophile to attack the phosphate, releasing tyrosine while forming a transient phosphocysteine intermediate (reviewed in refs. 14 and 15). The cysteine of the HCX 5 R motif located within the N-terminal domain of the Caenorhabditis elegans mRNA capping enzyme is required for its RNA triphosphatase activity, suggesting that phosphate hydrolysis occurs by a similar mechanism (1).The baculovirus protein phosphatase BVP (also known as BVH1 and BV-PTP) is a 19-kDa, PTP-like enzyme that can hydrolyze phosphoserine and phosphothreonine as well as phosphotyrosine residues (16-18). BVP exhibits 33% sequence identity to the N-terminal region (residues 1-174) of the CEL-1 capping enzyme triphosphatase (1, 2). Prompted by the high degree of sequence similarity to CEL-1, we tested BVP f...