In eukaryotic cells, gene expression involves multi-step processes in the nucleus and the cytoplasm. The different processes engage specific RNA-protein complexes, RNPs. Upon activation of most, if not all genes, a precursor RNA molecule is synthesized that has to be extensively processed and modified. In addition, the RNA has to associate with a distinct set of proteins. The composition of the RNP is often dynamic and changes over time. Several RNPs are exported to the cytoplasm, where they are involved in late steps of gene expression, including the synthesis of proteins.
The aim of this thesis has been to increase our knowledge about specific steps in messenger RNP (mRNP) and ribosomal RNP (rRNP) biogenesis in the eukaryotic cell.
We have characterized a novel protein, RBD-1, that is essential for ribosome biogenesis. RBD-1 contains six RNA-binding domains and is conserved in eukaryotes. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) is mainly located in the nucleolus, in an RNA polymerase I transcription-dependent manner. In cytoplasmic extracts, Ct-RBD-1 is preferentially associated with 40S ribosomal subunits. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress prerRNA processing in vivo. RBD-1 is essential in Caenorhabditis elegans. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis.
We have also studied the putative homologue to RBD-1 in Saccharomyces cerevisiae, Mrd1p. Mrd1p is essential for viability in yeast. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and a decrease in the steady-state level of 40S ribosomal subunits. Mrd1p associates with prerRNA and U3 snoRNA and is required for the initial cleavages of the pre-rRNA. It is likely that Mrd1p is involved in the structural coordination of the pre-rRNA during early processing steps.
We have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. We have studied its location and its relation to the transcription and translation processes. In the cytoplasm, Ct-eIF4H is associated with mRNA in polysomes. A minor fraction of CteIF4H is present in the nucleus, but it could not be detected in pre-mRNPs or mRNPs. The nuclear amount of Ct-eIF4H is independent of the level of transcription. We have addressed the question of where the translation machinery associates with mRNAs. Using immunoelectron microscopy, we can show that Ct-eIF4H associates with mRNPs in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.
Ct-RSF was isolated in a screen for RNA-binding proteins in Chironomus tentans. Ct-RSF has several properties in common with SR proteins, a family of conserved splicing factors. We have shown that Ct-RSF interacts with SR proteins, but in contrast to the splicing factors, Ct-RSF represses splicing in vitro. Our data suggest that Ct-RSF binds to exon sequences co-transcriptionally in vivo and that it represses the activation of splicing by SR proteins. It is conceivable that Ct-RSF is a protein that balances the action of SR proteins and avoids the formation of spliceosomes at aberrant splice sites in exons.
Finally, we have initiated a study of Ct-Y14 and Ct-Mago. Pre-mRNA splicing deposits a multi-protein complex 20-24 nucleotides upstream of exon-exon junctions. This complex, the EJC, is believed to couple the splicing process to nuclear export, nonsense-mediated decay and cytoplasmic localization of mRNAs. The EJC contains a number of proteins, including Y14 and Mago. To contribute knowledge about the in vivo dynamics of Y14 and Mago, we decided to analyze the association of Y14 and Mago with the BR mRNAs in Chironomus tentans.
Stockholm: Dep. of Molecular Biology and Functional Genomics , 2003. , 67 p.
2003-10-03, Sal G, Arrheniuslaboratorierna, Frescati, Stockholm, 10:00 (English)