mRNA Capping Enzyme Activity Is Coupled to an Early Transcription Elongation

Abstract

One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientationdependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.The eukaryotic mRNAs produced by RNA polymerase II (Pol II) are capped with an inverted 7-methyl-guanosine (m 7 G) linked to the first residue of the mRNA (36). This event 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 the diphosphate end. RNA guanylyltransferase forms a covalent enzyme-GMP complex and subsequently caps the RNA substrate by adding a guanosine residue in a 5Ј-5Ј triphosphate linkage. The cap is then methylated by RNA (guanine-7) methyltransferase (23, 39). In higher eukaryotes, a bifunctional monomeric polypeptide carries both the RNA triphosphatase and guanylyltransferase activities, while the capping enzyme from yeast is a complex of RNA triphosphatase and guanylyltransferase subunits (40). The polypeptides are encoded by the CET1 and CEG1 genes, respectively in Saccharomyces cerevisiae, and both are essential for the cell viability.Capping, the first mRNA modification, occurs by the time the transcript is only 25 to 30 nucleotides long in an early transcription phase. Such cotranscriptional capping is mediated by recruitment of capping enzyme machinery to the phosphorylated carboxy-terminal domain (CTD) of the largest subunit of Pol II (7,15,22,51). The CTD of Pol II has an unusual structure with many heptapeptide repeats (YSPTSPS). The capping enzyme binds directly and specifically to the phosphorylated CTD of Pol II via the Ceg1 subunit (yeast) or the guanylyltransferase domain (metazoan). Furthermore, the guanylyltransferase activity of Ceg1 associated with phosphorylated CTD is allosterically regulated by both the Cet1 and phosphorylated CTD to ensure it has a coordin...

Functional integration of transcriptional and RNA processing machineries

“…It was observed earlier that an excess amount of the methylation donor SAM inhibited transcription initiation [64] and that the 5′ capping enzymes were able to repress transcription [65], suggesting that the 5′ capping reaction and the subsequent release of the capping enzymes are both critical for the transition from transcriptional initiation to elongation. Indeed, further studies revealed that both Ser5 phosphorylated CTD and the transcription elongation factor Spt5 help recruit the 5′ capping enzymes, which then counteract the inhibitory NELF complex, thereby facilitating promoter clearance and enhancing the entrance of the initially paused RNAPII to the mode of productive transcription elongation [66][67][68]. After this transition, the CTD phosphatase FCP1 seems responsible for removing Ser5 phosphates and dismantling the capping enzymes [20,68].…”

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

“…For instance, stimulation of CE by HIV Tat promotes read-through of the viral genome (20,21). CE has been implicated in the formation of an early elongation checkpoint, as has been observed in 5Ј-paused Pol II complexes (15,(22)(23)(24)(25). Cotranscriptional capping is effectuated by the specific interaction between a CE and its cognate RNA polymerase that supports a stoichiometric CE-polymerase complex (13,26), as exemplified in certain viral transcription/processing systems in each of which the transcription of viral genes is mediated by a single virus-encoded RNA polymerase (27).…”

Human capping enzyme promotes formation of transcriptional R loops in vitro

“…Thus, CE-dependent RLF in yeast would only occur, if at all, at an early phase of transcription. Indeed, a ceg1 mutant strain with diminished GTase activity showed reduced Pol II elongation at promoterproximal regions (18). It is conceivable that this process in some way involves RLF.…”

“…More recent studies have shown that CE can act as a transcriptional regulator. In vitro experiments in yeast demonstrated that Cet1 represses transcription reinitiation (17), whereas mutation of ceg1 and abd1 affects transcription elongation (18,19). Finally, human CE is capable of relieving transcription repression by the negative elongation factor (NELF) (20).…”