Disease relevance of CEG1

• To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli [1].
• The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases [1].

High impact information on CEG1

• Ceg1 is released early in elongation, but Abd1 can travel with transcribing pol II as far as the 3' end of a gene [2].
• In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1) [3].
• We performed a structure-function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase [4].
• 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 [5].
• Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important [6].

Biological context of CEG1

• ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3' gene of unknown function previously sequenced (Shibagaki et al., 1992) [7].
• The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes [8].
• CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis [9].
• We have isolated 10 recessive, temperature-sensitive mutations of CEG1; nine (ceg1-1 to ceg1-9) were isolated on a single-copy plasmid and the remaining one (ceg1-10) on a multicopy plasmid [10].
• We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528] [11].

Associations of CEG1 with chemical compounds

• Plasmid shuffling experiment with Saccharomyces cerevisiae GTase subunit gene CEG1 null mutant demonstrated that deletion mutants 211-567 and 211-585 were able to support cell viability in the presence of 5-fluoroorotic acid, whereas 211-537 and 211-560 were not [12].
• Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates [13].
• Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities [14].
• It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed [14].

Physical interactions of CEG1

• Inhibition of mRNA turnover in yeast by an xrn1 mutation enhances the requirement for eIF4E binding to eIF4G and for proper capping of transcripts by Ceg1p [15].
• In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase [16].

Other interactions of CEG1

• These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity [17].
• Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not [15].
• We have isolated three Saccharomyces cerevisiae genes-CES1, CES2, and CES3-- that, when present in high copy, suppress the ts growth defect caused by mutations in the CEG1 gene encoding mRNA guanylyltransferase (capping enzyme) [18].
• Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants [19].

Analytical, diagnostic and therapeutic context of CEG1

• CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli [11].
• 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 degrees C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures [20].

References