Nuclear translation

Thesis

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute.

I also collaborated on two additional projects: the purification of mega-complexes (transcription 'factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).

Authors: Baboo, S

• Publication date: 2012
• DOI: 10.5287/ora-vjwxyjowq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: immuno-RNA FISH; proximity ligation assay; nonsense-mediated mRNA decay; L-azidohomoalanine; Copper-catalyzed azide-alkyne cycloaddition; intronic translation; puromycin; nuclear protein synthesis; click chemistry; CD2; Nuclear translation; antibody blocking; transcription factory
• Subjects: Biology; Protein chemistry; Microscopy; Cell Biology; Synthetic organic chemistry