De, Sandip (2011)
Ph.D. thesis, University of Birmingham.
Translation factors are essential components of the ribosome, yet it has been reported that many ribosomal proteins (RPs) and other translation factors are found at transcription sites of Drosophila melanogaster (D. melanogaster) polytene chromosomes. Whilst these findings might indicate the presence of ribosomal subunits at transcription sites, it has also been reported that these proteins associate with non-coding RNA in Saccharomyces cerevisiae (S. cerevisiae), suggesting that their localization with transcription sites reflects their non-ribosomal function. However, the functional significance of RPs and translation factors at transcription sites is unclear and may reflect excess protein synthesize unincorporated into ribosomes, leading to a large pool of free proteins able to interact non specifically with other proteins and nucleic acids. The following work investigates these issues further in Schizosaccharomyces pombe (S. pombe). I tagged three RPs (RpL7, RpL11 and RpL25), by homologous recombination and used a Chromatin immunoprecipitation approach to investigate whether the association of RPs occurs across specific genes/transcripts or whether chromatin association is genome wide. In agreement with previous studies in D. melanogaster and S. cerevisiae, I found that RPs preferentially associate to transcriptionally active genes. ChIP followed by analysis on micro-arrays (ChIP-on-chip) revealed that RPs associate with several protein encoding genes. Further analysis of the three RPs showed that they tend to bind a common subset of genes. Whilst RNase sensitivity suggests RPs association with nascent RNA, I found no correlation between the ChIP-on-chip signals of RPs with either Pol II occupancy or transcript level but did show that RPs associate with non-coding-RNA genes most notably with tRNA genes. ChIP of RpL7 in a strain carrying an exogenous wild-type tDNATyr gene or a mutant with a non-functional promoter confirmed that RpL7 associates only to the active tRNA gene. These results suggest a functional role of RPs in tRNAs biogenesis and perhaps in a role in Pol III transcription, as it was recently suggested by the finding that RPs copurify with TFIIIE in S. cerevisiae. Nonsense-mediated mRNA decay (NMD) removes any mRNA containing premature termination codon (PTC), and requires Upf1. Phospho-Upf1 inhibits conversion of 40S/Met-tRNAiMet/mRNA to translationally competent 80S/Met-tRNAiMet/mRNA initiation complexes to repress continued translation initiation. Analysis with Upf1 showed Upf1 also associates with many transcription sites and has a role in DNA replication and/or repair. Notably, Upf1 binds to chromatin mostly during S phase; perhaps indicating a role for its helicase activity during DNA replication in S. pombe. In summary, my data indicate that association of RPs, and at least one NMD factor, to chromatin is a general feature of eukaryotes. The main challenge for future studies is to identify the factors driving this association and the functional significance.
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