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Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis
Abstract:
To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.
Insights
Nucleolar phosphoprotein B23 is crucial for ribosome synthesis. Inhibiting ribosomal RNA processing causes B23 to relocate from nucleoli to the nucleoplasm, indicating its involvement in ribosome biogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nucleolar phosphoprotein B23 is implicated in ribosome biogenesis.
- Understanding its precise role requires investigating its localization during ribosomal RNA processing.
Purpose of the Study:
- To elucidate the role of nucleolar phosphoprotein B23 in ribosome synthesis.
- To investigate the translocation of protein B23 in response to drugs affecting ribosomal RNA processing.
Main Methods:
- Treatment of cells with drugs inhibiting ribosomal RNA processing (actinomycin D, toyocamycin, alpha-amanitin).
- Analysis of protein B23 localization using fluorescence microscopy.
- Quantification of protein B23 levels in nucleoli and nucleoplasm via ELISA immunoassay.
- Isolation and fractionation of preribosomal ribonucleoprotein particles (pre-rRNPs) from Novikoff hepatoma ascites cells.
- Analysis of pre-rRNPs using sucrose density gradients, gel electrophoresis, and immunoblotting.
Main Results:
- Inhibitors of ribosomal RNA processing caused uniform nucleoplasmic fluorescence of protein B23.
- Actinomycin D treatment led to a 60% decrease in nucleolar protein B23 and a 3-fold increase in nucleoplasmic protein B23.
- Protein B23 was co-localized with pre-ribosomal ribonucleoprotein particles (pre-rRNPs).
- Protein B23 was detected in both 80 S and 55 S pre-rRNPs.
Conclusions:
- Nucleolar phosphoprotein B23 translocates from the nucleolus to the nucleoplasm upon inhibition of ribosomal RNA processing.
- Protein B23 is associated with preribosomal ribonucleoprotein particles, supporting its role in ribosome synthesis.