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The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for
Toshiko Yamada-Okabe1, Toshiyuki Mio2, Yuji Kashima1
1Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan1.
Abstract:
The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1delta null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.
Insights
Researchers identified the Candida albicans ABD1 (CaABD1) gene, encoding an active RNA (guanine-N7-)-methyltransferase (cap MTase). Key leucine residues in a conserved motif are crucial for CaAbd1p
Area of Science:
- Molecular Biology
- Enzymology
- Fungal Genetics
Background:
- Eukaryotic mRNA 5'-cap methylation is vital for gene expression, catalyzed by RNA (guanine-N7-)-methyltransferases (cap MTases).
- The Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (CMT1) genes encode known cap MTases.
- Understanding fungal cap MTases is crucial for studying pathogenic fungi like Candida albicans.
Purpose of the Study:
- To clone and characterize the ABD1 homologue from Candida albicans (CaABD1).
- To determine if CaABD1 encodes a functional cap MTase.
- To investigate the functional importance of a conserved amino acid motif in CaAbd1p.
Main Methods:
- Cloning of the CaABD1 gene from Candida albicans.
- Expression of CaAbd1p as a glutathione S-transferase (GST) fusion protein.
- In vitro cap MTase activity assays.
- Complementation of Saccharomyces cerevisiae abd1delta null mutants.
- Yeast two-hybrid analysis to assess protein interactions.
- Site-directed mutagenesis of conserved amino acid residues within the cap MTase motif.
Main Results:
- CaABD1 was cloned and expressed; the resulting CaAbd1p displayed in vitro cap MTase activity.
- CaAbd1p functionally complemented S. cerevisiae abd1delta null mutants, confirming its role as an active cap MTase.
- Yeast two-hybrid analysis indicated no physical interaction between CaAbd1p and yeast mRNA guanylyltransferase or mRNA 5'-triphosphatase.
- Mutations of leucine at positions 2 and 12 within a conserved motif severely impaired CaAbd1p activity and in vivo function.
- Valine substitution at position 12 abolished CaAbd1p activity, suggesting side chain length is critical.
Conclusions:
- Candida albicans possesses a functional RNA (guanine-N7-)-methyltransferase encoded by CaABD1.
- CaAbd1p does not appear to physically interact with yeast capping enzymes.
- Specific leucine residues (positions 2 and 12) within a conserved methyltransferase motif are essential for CaAbd1p catalytic activity and function.