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Methylated simian virus 40-specific RNA from nuclei and cytoplasm of infected BSC-1 cells
Abstract:
Host cell and virus-specific poly(A)-containing RNAs isolated from nuclei and cytoplasm of monkey kidney cells infected with simian virus 40 contain different methylated nucleotides. In the cytoplasmic simian virus 40-specific RNA, about 75% of the radioactivity derived from (methyl-3-H)methionine was in N-6-methyladenosine (N-6mA) after digestion with Penicillium nuclease and bacterial alkaline phosphatase. The remainder was in a negatively charge component with properties of 5'-terminal structures, i.e., digestion with nucleotide pyrophosphatase and bacterial alkaline phosphatase released 2'-O-methyladenosine (A-m), 2'-O-methylguanosine (G-m), and 7-methylguanosine (m-7-G), consistent with a 5'-terminal structure of the type, m7-GpppNm. The nuclear virus-specific RNA contained N6mA, GM, 2'-O-methyluridine (U-m), and a smaller proportion (10%) of nuclease-, phosphatase-resistant presumptive 5' termini that also yielded A-m, G-m, and m7-G upon further hydrolysis. The infected cell nuclear and cytoplasmic RNAs that did not hybridize to DNA of simian virus 40 contained all four 2'-O-methylnucleosides. The possible role of methylation in the processing and translation of simian virus 40-specific mRNA is discussed.
Insights
Simian virus 40 infection alters methylated nucleotide composition in host cell RNA. Cytoplasmic viral RNA primarily features N-6-methyladenosine, while nuclear RNA shows diverse methylation patterns, impacting mRNA processing.
Area of Science:
- Molecular Virology
- RNA Biology
- Post-transcriptional Modification
Background:
- Simian virus 40 (SV40) is a DNA virus that infects monkey kidney cells.
- RNA processing, including methylation, plays a crucial role in gene expression.
- Understanding viral RNA modification is key to deciphering host-pathogen interactions.
Purpose of the Study:
- To investigate and compare methylated nucleotide content in host cell and SV40-specific RNAs.
- To analyze the distribution of methylated nucleotides in nuclear versus cytoplasmic fractions.
- To explore the potential role of RNA methylation in SV40 mRNA processing and translation.
Main Methods:
- Isolation of poly(A)-containing RNAs from SV40-infected monkey kidney cells (nuclei and cytoplasm).
- Radiolabeling with (methyl-3-H)methionine to trace methylation.
- Enzymatic digestion (Penicillium nuclease, bacterial alkaline phosphatase, nucleotide pyrophosphatase) followed by analysis of methylated nucleotides.
Main Results:
- Cytoplasmic SV40 RNA predominantly contains N-6-methyladenosine (N-6mA) (approx. 75%).
- A smaller fraction of cytoplasmic RNA exhibits 5'-terminal methylated structures (m7-GpppNm), yielding 2'-O-methyladenosine (A-m), 2'-O-methylguanosine (G-m), and 7-methylguanosine (m-7-G).
- Nuclear SV40 RNA shows a more diverse methylation profile, including N-6mA, G-m, 2'-O-methyluridine (U-m), and 5'-terminal structures (approx. 10%).
- Uninfected cellular RNAs contained all four 2'-O-methylnucleosides.
Conclusions:
- Significant differences exist in methylation patterns between nuclear and cytoplasmic SV40-specific RNAs.
- N-6mA is a major methylation site in cytoplasmic SV40 RNA, potentially influencing stability or translation.
- The observed methylation patterns suggest a role for these modifications in SV40 mRNA maturation and function.