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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

Parallel time-course assays measured with SPA and filter binding methods. Maroon are RmtA + wild-type 30S; orange are KsgA and ksgR 30S. Solid symbols indicate signal reactions (10 pmol 30S, 10 pmol enzyme, 20 μM SAM 780 cpm/pmol); empty symbols indicate background reactions (10 pmol 30S, 20 μM SAM 780 cpm/pmol). Measurements by YSi SPA beads (closed triangle) and the corresponding filter binding measurements (closed square). Assays were done in triplicate, plotting the average with error bars of ± 1 SD.
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Figure 2: Parallel time-course assays measured with SPA and filter binding methods. Maroon are RmtA + wild-type 30S; orange are KsgA and ksgR 30S. Solid symbols indicate signal reactions (10 pmol 30S, 10 pmol enzyme, 20 μM SAM 780 cpm/pmol); empty symbols indicate background reactions (10 pmol 30S, 20 μM SAM 780 cpm/pmol). Measurements by YSi SPA beads (closed triangle) and the corresponding filter binding measurements (closed square). Assays were done in triplicate, plotting the average with error bars of ± 1 SD.

Mentions: Parallel time course assays (Figure 2) were done as above except the reaction volume was sufficient for 16 samples for signal reactions and 6 samples for background reactions. Samples of 100 μl were removed at each time point, quenched with 20 μl 100 mM unlabeled SAM, and divided into two 60 μl aliquots for determination by the SPA and filter binding methods. Signal reactions were measured at 1, 2, 4, 8, 16, 32, 64 and 128 min, while background reactions were measured at 1, 8 and 128 min, all in triplicate.Figure 2.


Scintillation proximity assay for measurement of RNA methylation.

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

Parallel time-course assays measured with SPA and filter binding methods. Maroon are RmtA + wild-type 30S; orange are KsgA and ksgR 30S. Solid symbols indicate signal reactions (10 pmol 30S, 10 pmol enzyme, 20 μM SAM 780 cpm/pmol); empty symbols indicate background reactions (10 pmol 30S, 20 μM SAM 780 cpm/pmol). Measurements by YSi SPA beads (closed triangle) and the corresponding filter binding measurements (closed square). Assays were done in triplicate, plotting the average with error bars of ± 1 SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Parallel time-course assays measured with SPA and filter binding methods. Maroon are RmtA + wild-type 30S; orange are KsgA and ksgR 30S. Solid symbols indicate signal reactions (10 pmol 30S, 10 pmol enzyme, 20 μM SAM 780 cpm/pmol); empty symbols indicate background reactions (10 pmol 30S, 20 μM SAM 780 cpm/pmol). Measurements by YSi SPA beads (closed triangle) and the corresponding filter binding measurements (closed square). Assays were done in triplicate, plotting the average with error bars of ± 1 SD.
Mentions: Parallel time course assays (Figure 2) were done as above except the reaction volume was sufficient for 16 samples for signal reactions and 6 samples for background reactions. Samples of 100 μl were removed at each time point, quenched with 20 μl 100 mM unlabeled SAM, and divided into two 60 μl aliquots for determination by the SPA and filter binding methods. Signal reactions were measured at 1, 2, 4, 8, 16, 32, 64 and 128 min, while background reactions were measured at 1, 8 and 128 min, all in triplicate.Figure 2.

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