An Essential Function of Saccharomyces cerevisiae RNA Triphosphatase Cet1 Is to Stabilize RNA Guanylyltransferase Ceg1 against Thermal Inactivation

Abstract

Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35°C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures. Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265). In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265). We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269 -549)) provided that Ceg1 is also overexpressed. However, such cells are unable to grow at 37°C. In contrast, cells overexpressing Cet1(269 -549) in single copy grow at all temperatures if they express either the S. pombe or mammalian guanylyltransferase in lieu of Ceg1. Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase. We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity. We used protein-affinity chromatography to identify the COOHterminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain.

mRNA Capping Enzyme Requirement forCaenorhabditis elegans Viability

“…The inactive GTase mutant (N ϩ C K311A) did not complement in the ⌬Ceg1 strain. It also failed in ⌬Cet1, in agreement with CEG1 requiring CET1 for allosteric (25) and/or stabilizing (26) interactions.…”

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

“…Interestingly, a similar stimulation of the GTase (Ceg1) upon binding to the RTase (Cet1) is also observed in Saccharomyces cerevisiae, where the Cet1-Ceg1 interaction increases the extent of formation of the Ceg1-GMP complex (Ho et al 1998a(Ho et al , 1999Hausmann et al 2001). This interaction is crucial for the stabilization of the yeast GTase, which is inherently thermolabile (Hausmann et al 2001). Moreover, the interaction also enhances the affinity of the enzyme for the GTP substrate (Ho et al 1998b).…”

Homodimeric Quaternary Structure Is Required for the in Vivo Function and Thermal Stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA Triphosphatases

“…Cet1 by itself does not interact with the CTD. In contrast, the S. pombe guanylyltransferase Pce1 is inherently thermostable, and its stability is unaffected by the presence of the triphosphatase Pct1 (9). Also, S. pombe employs a distinctive strategy of targeting capping to polymerase II transcripts, whereby the Pct1 and Pce1 enzymes bind independently to the phosphorylated CTD (8).…”

“…The Cet1-Ceg1 interaction stabilizes the intrinsically labile guanylyltransferase activity of Ceg1 against thermal inactivation at physiological temperatures (9). In addition, the physical tethering of Cet1 to Ceg1 facilitates recruitment of the triphosphatase to the RNA polymerase II elongation complex, via Ceg1 binding to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II (10 -13).…”

“…However, the monomeric domain by itself cannot support yeast cell growth, even when it is overexpressed at high gene dosage under the control of a strong promoter. Interpretation of the deletion data is complicated by the fact that an N-terminal truncation to position 275 also removes the guanylyltransferase-binding site 247 WAQKW 251 , which is located on the protein surface (4,16,20,21) and is responsible for Cet1-mediated stabilization of the guanylyltransferase Ceg1 (9). The in vivo function of Cet1(276 -549) is restored when the monomeric triphosphatase is fused to either S. pombe guanylyltransferase (Pce1) or the guanylyltransferase domain of mammalian capping enzyme (4,9).…”