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Published on: September 10, 2015
Transcript localization in Dictyostelium discoideum cells by RNA FISH
Patrick Hofmann1, Janis Kruse, Christian Hammann
1Heisenberg Research Group Ribogenetics, Technical University of Darmstadt, Darmstadt, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2013
Summary
Ribosomal RNA (rRNA) biogenesis involves precursor processing. This study adapted fluorescence in situ hybridization (FISH) to track rRNA molecules in Dictyostelium discoideum cells, localizing them to the nucleolus and cytoplasm.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Ribosomal RNA (rRNA) biogenesis initiates in the nucleolus, the site of ribosomal DNA (rDNA) transcription in eukaryotic cells.
- Evidence suggests that certain rRNA processing steps occur in the cytoplasm, but direct visualization has been challenging.
Purpose of the Study:
- To adapt and apply fluorescence in situ hybridization (FISH) protocols for visualizing rRNA processing events in Dictyostelium discoideum.
- To generate specific fluorescently labeled probes for different rRNA molecules.
- To determine the subcellular localization of precursor and mature rRNA during biogenesis.
Main Methods:
- Development of protocols for cell fixation suitable for FISH in Dictyostelium discoideum.
- Synthesis and labeling of oligonucleotide probes targeting specific rRNA sequences.
- Application of FISH to detect and localize rRNA precursors and mature rRNA within the cell.
Main Results:
- Successful localization of precursor rRNA molecules to the nucleolus.
- Localization of mature rRNA molecules to the cytoplasm.
- Demonstration of the utility of FISH for studying rRNA biogenesis in Dictyostelium.
Conclusions:
- The adapted FISH technique effectively visualizes rRNA localization during biogenesis in Dictyostelium.
- Precursor rRNA is primarily found in the nucleolus, while mature rRNA is localized to the cytoplasm.
- This study provides a valuable tool for investigating rRNA processing and localization in this model organism.
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