Proteomics and dynamics of the human nucleolus

Abstract

The nucleolus is the most prominent structure within the eukaryotic cell nucleus and it was established to be the site where the majority of ribosomal RNAs (5.8S, 18S and 28S) are transcribed, processed and assembled with ribosomal proteins to form ribosomal subunits. The sole role of ribosome biogenesis, however, cannot explain the specific nucleolar localisations of tumour suppressors, cell cycle-regulatory factors and viral proteins. Therefore, together with my colleagues in the laboratory of Prof. Angus Lamond, we carried out a proteomic approach with an aim to identify the core components of the human nucleolus isolated from HeLa cell nuclei. My role in this project includes verification of the newly identified components, database construction archiving the primary data and providing links to other related information in the public domain, and subsequent bioinformatics and microscopic analyses. So far, 400 proteins were identified in which -30% represents novel or uncharacterised proteins, partly reflecting the current poor status in the human genome annotation, but also reflecting the unknown complexity of the nucleolus. To facilitate the understanding of the functions of these novel proteins, I used deposited
data of their gene activities and homologues across the species to identify in silico
those novel proteins that are likely to be involved in ribosomal biogenesis.
Like the nucleus, the nucleolus itself is subcompartmentalised into different domains, namely, the fibrillar centre, the dense fibrillar components and the granular components and these structures are disassembled and reassembled during mitosis in human cells. In order to understand the intricate mechanism behind these mitotic dynamics, I have generated a panel of 24 HeLa cell lines stably expressing one or more nucleolar marker to study the inter-relationships between these subnucleolar domains as well as their relationships with the chromosomes. The results suggest that (1) a core subunit of the RNA polymerase I dissociates from the chromosomes between prophase and metaphase and (2) the breakdown and reassembly are dependent on the dissociation and the recruitment of RNA polymerase I to the chromosomes respectively.As part of the follow-up to the nucleolar proteome identified, the study of one
uncharacterised factor NHPX led to the discovery of a novel nucleolar targeting
pathway that is observed in both primary and transformed cell lines. Although NHPX co-localises with the dense fibrillar component marker fibrillarin, NHPX transiently transits through the splicing speckles prior to the nucleolar accumulation whilst fibrillarin accumulates within the nucleolus immediately after the nuclear entry. The NHPX progression is dependent on pre-mRNA transcription and may link multiple RNA metabolic pathways that occur in distinct subnuclear domains.

Date of Award	2003
Original language	English
Supervisor	Angus Lamond (Supervisor)