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DNAzyme-dependent Analysis of rRNA 2’-O-Methylation
Published on: September 16, 2019
Methylation of ribonucleic acid in a cell-free system from mouse myeloma cells
Abstract:
Isolated nuclei incorporate few methyl groups into RNA when they are incubated with S-adenosyl[methyl-3H3]methionine and four ribotriphosphates. When the nuclei were supplemented with a soluble total cell protein extract, the incorporation of methyl groups into RNA was stimulated 3-6-fold. All classes of RNA were methylated. Methylation of the 2'-OH of ribose and the bases of ribosomal RNA occurred predominantly on endogenous ribosomal RNA precursors, with a minority (20%) occurring on the newly synthesized rRNA precursor. Methylation of the tRNA precursor occurred on both endogenous (40%) and newly synthesized (60%) molecules. The methylation of adenosine in hnRNA occurred predominantly on molecules transcribed in vitro and was sensitive to 1 microgram/mL alpha-amanitin. A final site of methylation was the 7 position of guanosine of the cap structure. About 10% of the RNA polymerase II transcripts were capped in vitro. Capping was blocked 90% by 1 microgram/mL alpha-amanitin and was independent of the presence of the cell protein extract.
Insights
Cell extracts significantly boost RNA methylation in isolated nuclei, affecting ribosomal RNA, tRNA, and hnRNA. Methylation occurs on both existing and newly synthesized RNA molecules, including RNA polymerase II transcripts.
Area of Science:
- Molecular Biology
- Biochemistry
- Gene Expression
Background:
- RNA methylation is crucial for RNA processing and function.
- Isolated nuclei have limited capacity for RNA methylation.
- Cellular factors are known to influence RNA modification.
Purpose of the Study:
- To investigate the role of soluble cell protein extracts in RNA methylation within isolated nuclei.
- To identify the specific classes of RNA and modification sites affected by these extracts.
- To elucidate the mechanisms regulating RNA methylation in vitro.
Main Methods:
- Incubation of isolated nuclei with S-adenosyl[methyl-3H3]methionine and ribotriphosphates.
- Supplementation with soluble total cell protein extract.
- Analysis of RNA methylation across different RNA classes (rRNA, tRNA, hnRNA) using radiolabeling.
- Assessment of alpha-amanitin sensitivity for RNA polymerase II transcripts.
Main Results:
- Soluble cell protein extract stimulated RNA methylation 3-6 fold in isolated nuclei.
- All major RNA classes (rRNA, tRNA, hnRNA) showed increased methylation.
- Methylation occurred on both endogenous precursors and newly synthesized RNA molecules.
- RNA polymerase II transcripts exhibited N7-guanosine capping, sensitive to alpha-amanitin.
Conclusions:
- Soluble cell protein extracts contain factors essential for efficient RNA methylation in vitro.
- Nuclear RNA methylation involves modifications on both pre-existing and newly transcribed RNA.
- Specific methylation sites, including 2'-OH ribose, base methylation, and N7-guanosine capping, are regulated by cellular factors and transcription machinery.
