Abdullahi, Akilu Sada
(2015).
Visualisation of ribosomal subunits interaction reveals 80s ribosomes in the nucleus of Drosophila cells.
University of Birmingham.
Ph.D.
Abstract
In eukaryotes, transcription and RNA processing events are spatially separated from translation by the nuclear envelope. Although ribosomal subunits are synthesised and assembled in the nucleus, it is believed that they are kept inactive whilst in the nucleus. Yet, there were observations from this and other laboratories that suggest translation can occur in the nucleus. To further investigate whether translating ribosomes exist in the nucleus, I employed a bimolecular fluorescence complementation (BiFC) 80S reporter based technique previously developed in our laboratory that detects 80S assembly in \(Drosophila\) cells. The initial characterization indicated that the assay reports ribosomal subunit interaction, but other explanations could not be excluded. My first aim was to assess whether this technique is genuinely reporting translation-dependent subunits association. My results were consistent with the assay reporting 80S ribosomes formed as a consequence of translation. Following up from this, I developed a similar technique with Venus, which resulted in a more sensitive 80S reporter. While the 80S signal was more apparent in the cytoplasm, in both cell culture and fly tissue cells, a fraction of the cells showed a signal in the nucleus, particularly concentrated in the nucleolus. This signal was enhanced by translation elongation inhibitors in both cytoplasm and nucleus indicating that the detected 80S are engaged in translation. Notably, the nucleolar signal was prevented by RNA Pol II inhibition, suggesting that the 80S might be associated with mRNA in the nucleolus.
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