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.