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Scintillation proximity assay for measurement of RNA methylation.

Baker MR, Zarubica T, Wright HT, Rife JP - Nucleic Acids Res. (2009)

Bottom Line: Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time.In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA.We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298-0133, USA.

ABSTRACT
Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC'. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA-protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.

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Related in: MedlinePlus

Inhibitors of methylation. For each concentration of inhibitor, inhibitor/DMSO was added to 30 pmol RmtA + 30 pmol wild-type 30S ribosomal particles + 20 μM SAM 780 cpm/pmol for an 8 min reaction. One-third of the reaction volume was added to each of the three vials of SPA beads and the average plotted with error bars of ± 1 SD. (A) Reactions with sinefungin (closed circle) and with SAH (open circle). (B) Reactions with DMSO alone.
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Figure 7: Inhibitors of methylation. For each concentration of inhibitor, inhibitor/DMSO was added to 30 pmol RmtA + 30 pmol wild-type 30S ribosomal particles + 20 μM SAM 780 cpm/pmol for an 8 min reaction. One-third of the reaction volume was added to each of the three vials of SPA beads and the average plotted with error bars of ± 1 SD. (A) Reactions with sinefungin (closed circle) and with SAH (open circle). (B) Reactions with DMSO alone.

Mentions: Inhibition studies were done using RmtA and wild-type 30S in a triple volume reaction. Inhibitor or solvent was added at a specified concentration prior to the addition of SAM to initiate the reaction. Reactions were stopped after 8 min and dispensed in equal volumes into three vials with YSi beads. Sinefungin (Sigma) was dissolved in deionized H2O, SAH (Sigma) was dissolved in DMSO (American Bioanalytical). Data in Figure 7A and B were fitted with a sigmoidal dose–response curve provided in Sigma Plot 8.0; IC50 values were calculated as described (13).


Scintillation proximity assay for measurement of RNA methylation.

Baker MR, Zarubica T, Wright HT, Rife JP - Nucleic Acids Res. (2009)

Inhibitors of methylation. For each concentration of inhibitor, inhibitor/DMSO was added to 30 pmol RmtA + 30 pmol wild-type 30S ribosomal particles + 20 μM SAM 780 cpm/pmol for an 8 min reaction. One-third of the reaction volume was added to each of the three vials of SPA beads and the average plotted with error bars of ± 1 SD. (A) Reactions with sinefungin (closed circle) and with SAH (open circle). (B) Reactions with DMSO alone.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651799&req=5

Figure 7: Inhibitors of methylation. For each concentration of inhibitor, inhibitor/DMSO was added to 30 pmol RmtA + 30 pmol wild-type 30S ribosomal particles + 20 μM SAM 780 cpm/pmol for an 8 min reaction. One-third of the reaction volume was added to each of the three vials of SPA beads and the average plotted with error bars of ± 1 SD. (A) Reactions with sinefungin (closed circle) and with SAH (open circle). (B) Reactions with DMSO alone.
Mentions: Inhibition studies were done using RmtA and wild-type 30S in a triple volume reaction. Inhibitor or solvent was added at a specified concentration prior to the addition of SAM to initiate the reaction. Reactions were stopped after 8 min and dispensed in equal volumes into three vials with YSi beads. Sinefungin (Sigma) was dissolved in deionized H2O, SAH (Sigma) was dissolved in DMSO (American Bioanalytical). Data in Figure 7A and B were fitted with a sigmoidal dose–response curve provided in Sigma Plot 8.0; IC50 values were calculated as described (13).

Bottom Line: Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time.In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA.We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298-0133, USA.

ABSTRACT
Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC'. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA-protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.

Show MeSH
Related in: MedlinePlus