Al-Jubran, Khalid
(2010).
Molecular visualization and localization of ribosomal subunits interaction in cells.
University of Birmingham.
Ph.D.
Abstract
Eukaryotic cells are highly compartmentalized, so that some steps of gene expression occur in the nucleus and others in the cytoplasm, i.e., transcription and pre-mRNA processing are spatially separated from translation which occurs in the cytoplasm. Ribosome subunits and several translation factors are present in the nucleus but it is understood that they can interact to form the functional ribosome only during translation in the cytoplasm. Recent studies, however, have suggested that translation may occur also in the nucleus. That functional ribosomes may exist also in the nucleus has been suggested by studies in the field of nonsense mediated mRNA decay (NMD) and, more directly, by reports that ribosomal proteins are found associated with nascent transcripts on Drosophila polytene chromosome and that amino acids are incorporated at transcription sites. My project aimed to investigate further the question of whether there are functional ribosomes in the nucleus; and also to confirm the presence of ribosomal proteins at active transcription sites. To address these issues, I have developed a system to visualize ribosomal subunits interaction at the molecular level. The technique consists in tagging pair of ribosomal proteins, located at interaction surface of the 40S and 60S subunits, with split fragments of yellow fluorescent protein so that bimolecular fluorescence complementation (BiFC) occurs only when the subunits join to form a 80S ribosome. With this technique I was able to visualize translation sites in Drosophila S2 cells and in transgenic flies. Translation sites are most apparent in the cytoplasm, however in cells in which export of ribosomal subunits was blocked by drug treatment a clear signal is visible also in the nucleoplasm. Notably, I also I 2 detected a strong signal in the nucleolus. These observations suggest that either there is translation in the nucleolus or that, contrary to what is currently understood, ribosome subunits can join together during ribosome biogenesis. In summary, with the technique I have developed I was able to find further evidence that functional ribosomes are present in the nucleus. This technique and these first results shall aid future investigations into the fundamental issue of whether ribosomes have a function in the nucleus and also to monitor translation changes in living cells.
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