Investigating methods of visualising translation in schizosaccharomyces pombe

[摘要] Gene expression is compartmentalised in eukaryotes due to the nuclear envelope separating the nuclear processes of transcription and pre-mRNA processing from cytoplasmic translation. While ribosomes are synthesised in the nucleus, it is understood that a number of mechanisms keep them inactive until they reach the cytoplasm, where they mature to become translation-competent. However, this consensus view is being challenged by a growing body of evidence in support of nuclear translation. A newly developed technique, known as ribopuromycylation (RPM), had reported the presence of puromycin-bound nascent peptides on immobilised ribosomes in the nuclei of human cells. I investigated whether this method could be used, combined with chromatin immunoprecipitation, to determine whether nuclear ribosomes can cotranscriptionally translate nascent transcripts in Schizosaccharomycespombe. Surprisingly, I discovered that, in contrast to that reported in the original study, immobilising ribosomes with translation elongation inhibitors does not lead to retention of puromycylated peptides on ribosomes in either S. pombe,Drosophilamelanogaster or HeLa cells. However, I show here preliminary data which suggest that despite puromycylated peptides being released from the ribosome, puromycin immunostaining might still be used tovisualise the sub­ cellular localisation of ribosomes inS. pombe, along with other approaches which I also describe.

[发布机构] University:University of Birmingham;Department:School of Biosciences

[关键词] Q Science;QD Chemistry