Role of ubiquitin fusion ribosomal proteins in the synthesis and function of yeast ribosomes
- Autores: Antonio Fernández Pevida
- Directores de la Tesis: Jesús de la Cruz Díaz (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2014
- Idioma: inglés
- Número de páginas: 141
- Tribunal Calificador de la Tesis: Agustín Vioque Peña (presid.), Fernando Monje-Casas (secret.), Susana Rodríguez Navarro (voc.), Jerónimo Bravo Sicilia (voc.), Jorge Pérez Fernández (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Idus
- Resumen
All ubiquitin molecules in the cell are initially produced as precursor proteins. In yeast, there are 4 ubiquitin coding genes, named UBI1, UBI2, UBI3 and UBI4 (Ozkaynak et al, 1987). The first three genes encode for a linear ubiquitin fusion with distinct r-proteins, thus, these genes are also known as RPL40A/UBI1, RPL40B/UBI2 and RPS31/UBI3 (Finley et al, 1989). These genes provide cells with ubiquitin under exponential growth conditions. UBI4 encodes a 5 head-to-tail repeats of ubiquitin and has an important role under stationary phase and stress conditions. UBI1 and UBI2 are duplicated genes that encode for the L40 r-protein. These genes produce ubiquitin fused to two identical LSU r-proteins of 52 amino acids, L40A and L40B. The ubiquitin moiety is interrupted at the same position by one intron that is not homologous in each gene. On the other hand UBI3 is a non-essential gene that encodes for a ubiquitin fused to the SSU r-protein S31 of 76 amino acids (Finley et al, 1989).
L40 is a globular protein that assembles close to L12 at the base of the ribosomal stalk structure. S31 has a globular domain and an extension, but in contrast with other r-protein¿s extensions, this one stays at the surface rather than inside the structure. When both r-subunits join in an 80S particle, L40 and S31 are located opposed each other near the binding site of the elongation factors (Ben-Shem et al, 2011; Klinge et al, 2011). It has been proposed that the presence of the ubiquitin moieties within the r-proteins in the mature ribosome would prevent the proper association of the elongation factors. Thus, cleavage of ubiquitin will act as a mechanism of surveillance. However, this is only a speculation since the dynamics of ubiquitin processing still remain uncharacterised.
The aim of my PhD project consists in understanding the role of the r-proteins that are naturally fused to ubiquitin in the synthesis and function of yeast ribosomes. In S. cerevisiae, these proteins are L40 and S31.
To address this objective, we described the contribution of L40 in ribosome biogenesis. This study has been published as Fernandez-Pevida et al., 2012 (Fernández-Pevida et al, 2012).
We are also interested in revealing the role of ubiquitin in the 60S r-protein assembly process. For this purpose, we analyse cis-mutations that affect the cleavage of the ubiquitin.
Our group has previously studied the role of the ubiquitin moiety of the S31 r-protein in ribosome biogenesis (Lacombe et al, 2009). In the last part of my PhD project, we have studied the function of the eukaryotic-specific N-terminal extension of S31 in the assembly of 40S r-subunits. More specifically, we have analyzed how the deletion of this region, which contains a putative nuclear localization signal, affects ribosome biogenesis.
In this study, we have undertaken the functional analysis of L40 in yeast ribosome synthesis. Homologues of L40 are found in archaea (archaeal L40e) but not in bacteria (Nakao et al, 2004; Wu et al, 2008). The role of the ubiquitin moiety of yeast Ubi3 has been addressed by our group (Finley et al, 1989; Lacombe et al, 2009), however, that of Ubi1 and Ubi2 remains unexplored.
The polysome profile results, pulse-chase labelling, northern hybridisation and primer extension analyses in the L40-depleted strain indicate that L40 is practically dispensable for pre-rRNA processing. Thus, we could only observe a modest delay in 27SB and 7S pre-rRNA processing upon depletion of L40.
Polysome profile analyses after L40 depletion strongly indicate that 60S r-subunits lacking L40 are defective in subunit joining and suggest that both r-subunits may have an increased susceptibility to degradation at longer time points of L40 depletion; similar observations were made for the null rpl24 or null rpl29 mutants and upon L1 depletion (Baronas-Lowell & Warner, 1990; DeLabre et al, 2002; McIntosh et al, 2011; Pöll et al, 2009).
We present several lines of evidence to state that yeast L40 may assemble in the cytoplasm but, surprisingly, pre-60S particles are retained in the nucle(ol)us upon depletion of L40. Interestingly, our data demonstrate that L40 is an additional r-protein involved in the release of Nmd3 and Rlp24 from cytoplasmic pre-60S r-particles. Therefore, the nuclear accumulation of pre-60S upon L40 depletion is due to a lack of export factors in the nucleus that are hijacked in the cytoplasm.
In addition, we hypothesise that L40 participates in the translocation process during translation. This is deduced from the selective sordarin hypersensitivity of the rpl40a¿ and rpl40b¿ mutants and the increased resistance to this drug of cells expressing HA-tagged L40 as the sole source of L40.
We analyse cis-mutations that affect the cleavage of the ubiquitin in the Ubi1 precursor for revealing the role of ubiquitin in the 60S r-protein assembly process. Several mutations in the ubiquitin system have been previously studied in S. cerevisiae. For example, when the ubiquitin G76A variation (UbG76A) is expressed in a wild-type strain and the ubiquitylation machinery is inactivated, an accumulation of free polyubiquitin chains is detected because the deubiquitylation machinery cannot disassemble these chains (Hodgins et al, 1992). This leads to a phenotype similar to that found in situations of ubiquitin deprivation. The analysis of different artificial fusion proteins in S. cerevisiae, containing mutations within the N-terminal ubiquitin moiety, has been used to define the ubiquitin cleavage requirements (Butt et al, 1988; Johnson et al, 1992) and to the identification of the ubiquitin fusion degradation proteolytic pathway, which recognizes ubiquitin as a degradation signal (Johnson et al, 1995).
In a recent work involving the Ubi3 precursor, performed in collaboration with our group, Lacombe et al. analyzed several mutants to better understand the role of the ubiquitin moiety in the assembly of the r-protein S31 within 40S r-subunits and demonstrate that the cleavage of the ubiquitin is required for the efficient production and functional integrity of 40S ribosomal subunits.
In a part of the PhD project we address the role of the ubiquitin moiety in the assembly of L40 within 60S r-subunits. Only preliminary data has been generated for this project. To this end, we have generated and analysed, as previously done for UBI3, mutants where the efficiency of ubiquitin removal is reduced.
These mutations were ubi1G75A, ubi1G76A, ubi1G75,76A and ubi1+P77. As expected, all these mutations impair the cleavage of the ubiquitin. There is a direct correlation between growth defects and efficiency of cleavage. Therefore, the growth defect and precursor accumulation of ubi1G76A is much stronger than that of ubi1G75A. In the other hand, the mutations ubi1G75,76A and ubi1+P77 were lethal and completely abolish the processing. Polysome profile analysis of these mutants revealed that these mutations lead to a profile similar to those mutants that are affected in r-subunits joining and also, uncleaved precursors were only detected in the 60S fractions, suggesting that they are able to assemble into pre-60S r-particles that are not capable to engage into translation. Further experiments are necessary to better understand these effects.
The eukaryotic ribosome is larger than that of prokaryotes, containing an additional rRNA and additional r-proteins. Only 32 r-proteins out of the 79 eukaryotic r-proteins are common to both, eukaryotes and prokaryotes. Even, many of the eukaryotic r-proteins that are conserved in prokaryotes harbor large additional extensions that are eukaryote-specific (Lecompte et al, 2002).
The S31 r-protein consists of a C-terminal globular domain and an N-terminal extension. The specific roles of each domain have not been studied yet. In the last part of my PhD project, we aim to analyze the role of the N-terminal extension of the yeast S31 r-protein (Ben-Shem et al, 2011) by deletion of this extension of S31, hereafter ubi3¿N.
We have demonstrated that the N-terminal extension of S31 is necessary for cell growth and S31 stability. Last events of maturation of 18S rRNA are slightly affected in the ubi3¿N strain. This result is in agreement with the role of S31 in 20S pre-rRNA maturation, as shown by Lacombe et al.
The ubi3¿N mutant has a lesser decrease in 40S r-subunits than a null strain. Take into account that this truncated version of S31 still harbors its assembly capability. This suggests the presence of a mix population of two different 40S r-subunits in the ubi3¿N mutant, one lacking S31 and the other containing S31¿N.
The analysis of the S31 sequence by the NLS Mapper program, predicted that the S31 N-terminal extension contains a putative bipartite NLS. Our results clearly confirmed the functional relevance of this NLS sequence.
We look for S31 localization after blocking the pre-40S r-particles export. In these conditions, the S31-GFP is localized in the nucleus. Interestingly, the truncated version of S31 fused to GFP (S31¿N-GFP) is only present in the cytoplasm even for long time points of nuclear block. Therefore, the N-terminal extension is necessary for targeting S31 to the nucleus. Altogether, these experiments strongly suggest that S31 is assembled in the nucleus.
