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In vitro methylation of nascent reovirus mRNA by a virion-associated methyl transferase
Abstract:
Chymotrypsin-derived cores, but not virions, catalyze the transfer of methyl groups from S-adenosyl methionine (SAM) to nascent mRNA synthesized in vitro by the core polymerase. The reaction requires Mg(++), and is dependent on the presence of all 4 ribonucleoside triphosphates (rNTPs). Methylation proceeds optimally at 51 degrees C. All ten species of mRNA become methylated during transcription and it is estimated that one methyl residue is incorporated per RNA chain. Experiments designed to determine the location of the methylated nucleotide clearly demonstrate that methylation occurs exclusively at the 5' ends of nascent mRNA.
Insights
Chymotrypsin-derived cores catalyze mRNA methylation using S-adenosyl methionine (SAM). This process, essential for nascent RNA synthesis, occurs at the 5' end of mRNA molecules.
Area of Science:
- Molecular Biology
- Biochemistry
- Virology
Background:
- Core particles derived from chymotrypsin, not intact virions, possess enzymatic activity.
- The study investigates the role of these core particles in modifying newly synthesized RNA.
Purpose of the Study:
- To elucidate the mechanism of mRNA methylation by chymotrypsin-derived cores.
- To identify the factors and conditions necessary for this methylation reaction.
Main Methods:
- In vitro synthesis of nascent mRNA using core polymerase.
- Incubation with S-adenosyl methionine (SAM) and other necessary components.
- Analysis of methylated mRNA products to determine location and extent of methylation.
Main Results:
- Chymotrypsin-derived cores, but not virions, catalyze methyl group transfer from SAM to nascent mRNA.
- The reaction requires magnesium ions (Mg++), all four ribonucleoside triphosphates (rNTPs), and optimal temperature of 51°C.
- Methylation occurs exclusively at the 5' end of all ten mRNA species, with one methyl residue incorporated per RNA chain.
Conclusions:
- Chymotrypsin-derived core particles are responsible for the 5' end methylation of nascent mRNA.
- This methylation is an integral part of the in vitro transcription process, dependent on specific cofactors and conditions.