chaperone; AAA-ATPase; ribosome biogenesis; nuclear protein import; Saccharomyces cerevisiae; DEAD-box RNA helicase; ribosomal protein; yeast genetics; biochemistry; cell biology
Mitterer Valentin, Murat Guillaume, Rety Stephane, Blaud Magali, Delbos Lila, Stanborough Tamsyn, Bergler Helmut, Leulliot Nicolas, Kressler Dieter, Pertschy Brigitte (2016), Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation, in NATURE COMMUNICATIONS
, 7, 10336.
Fernandez-Pevida Antonio, Martin-Villanueva Sara, Murat Guillaume, Lacombe Thierry, Kressler Dieter, de la Cruz Jesus (2016), The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits, in NUCLEIC ACIDS RESEARCH
, 44(16), 7777-7791.
Pausch Patrick, Singh Ujjwala, Ahmed Yasar Luqman, Pillet Benjamin, Murat Guillaume, Altegoer Florian, Stier Gunter, Thoms Matthias, Hurt Ed, Sinning Irmgard, Bange Gert, Kressler Dieter (2015), Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones, in NATURE COMMUNICATIONS
, 6, 7494.
Rodriguez-Galan Olga, Garcia-Gomez Juan J., Kressler Dieter, de la Cruz Jesus (2015), Immature large ribosomal subunits containing the 7S pre-rRNA can engage in translation in Saccharomyces cerevisiae, in RNA BIOLOGY
, 12(8), 838-846.
Pillet Benjamin, Garcia-Gomez Juan J., Pausch Patrick, Falquet Laurent, Bange Gert, de la Cruz Jesus, Kressler Dieter (2015), The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site, in PLOS GENETICS
, 11(10), e1005565.
García-Gómez Juan J, Fernández-Pevida Antonio, Lebaron Simon, Rosado Iván V, Tollervey David, Kressler Dieter, de la Cruz Jesús (2014), Final pre-40S maturation depends on the functional integrity of the 60S subunit ribosomal protein L3., in PLoS genetics
, 10(3), 1004205-1004205.
Fernández-Pevida Antonio, Kressler Dieter, de la Cruz Jesús (2014), Processing of preribosomal RNA in Saccharomyces cerevisiae, in Wiley Interdiscip Rev RNA
, Epub ahead.
Pratte Dagmar, Singh Ujjwala, Murat Guillaume, Kressler Dieter (2013), Mak5 and Ebp2 act together on early pre-60S particles and their reduced functionality bypasses the requirement for the essential pre-60S factor Nsa1, in PLoS One
, 8(12), e82741.
The process of ribosome biogenesis is evolutionarily conserved among eukaryotes and it constitutes a main cellular activity. Most of our current knowledge concerning this highly dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae. The combined use of proteomic, genetic, and cell biological methods has revealed that a multitude of protein trans-acting factors (>200) are required for the assembly and maturation of pre-ribosomal particles as they travel from the nucleolus to the cytoplasm. Amongst these are, in agreement with the dynamic nature of the process, energy-consuming enzymes such as AAA-type ATPases, ATP-dependent RNA helicases and GTPases. This suggests that the energy derived from nucleotide hydrolysis confers directionality to ribosome assembly and that such a large number of trans-acting factors is required to ensure accurate and efficient synthesis of ribosomal subunits. However, the molecular mechanisms driving ribosome assembly remain largely elusive. Moreover, it has so far not been addressed how the protein building blocks of pre-ribosomal particles (ribosomal proteins and trans-acting factors) reach their site of assembly in the nucle(ol)us. Therefore, current challenges in the field consist in the identification of the specific substrates of energy-consuming enzymes and in the understanding of their mechanism and timing of action. Furthermore, it will be of importance to attribute a functional role to each protein trans-acting factor involved in ribosome biogenesis. In order to understand the process at a molecular level, it will be necessary to gain structural insight into these factors and the pre-ribosomal particles that they are associated with and acting on. It is also not understood how the sequential action of these factors is coordinated, hence, the regulation of individual biogenesis steps has to be addressed. Finally, we will have to understand how ribosomal proteins contribute to ribosome biogenesis and how ribosomal proteins and trans-acting factors travel from their site of cytoplasmic synthesis to their mostly nucle(ol)ar assembly site. The aim of this proposal is to provide molecular insight into selected aspects of eukaryotic ribosome biogenesis. To this end, we will use S. cerevisiae as a model system, which is especially amenable to the application of a wide variety of biochemical, cell biological and unique genetic methods.Specifically, I propose the following projects:1) Analysis of the Nsa1/pre-ribosome interactionWe have previously shown that the AAA-type ATPase Rix7 is required for the release of Nsa1 from pre-60S ribosomal particles, thereby triggering the progression of 60S biogenesis. Furthermore, we have recently solved the crystal structure of Nsa1, which forms a 7-bladed WD-repeat ß-propeller. Here, we will exploit the Nsa1 crystal structure in order to identify by mutational analysis the surface on Nsa1 that mediates binding to the pre-60S ribosome. In a complementary approach, we will determine by electron microscopy the binding site of Nsa1 on pre-60S ribosomes.2) Co-translational recruitment of ‘chaperones’ to nascent ribosomal proteinsRecent evidence suggests that certain ribosomal proteins require distinct ‘chaperone’ proteins in order to be stably expressed and delivered to their site of assembly. We have shown for a set of four ribosomal proteins (Rps3, Rpl3, Rpl5, and Rpl10) that their ‘chaperone’ partners (Yar1, Rrb1, Syo1, and Sqt1) recognize the very N-terminal residues, notably implicated in rRNA binding, of the ribosomal proteins. Therefore, we aim to demonstrate that these ‘chaperones’ are recruited to nascent ribosomal proteins, thus establishing a novel step of ribosome assembly, commencing with the recognition of ribosomal proteins during translation. We will complement this study by structural analyses of the ‘chaperone’/ribosomal protein interactions.3) Nuclear co-import of the Enp1-Ltv1-Rps3-Yar1 complexOur recent study on the synchronization of nuclear import and assembly of two ribosomal proteins by the transport adaptor Syo1, suggests that nuclear import of functional units or pre-assembled (sub-)complexes may represent a general strategy to streamline downstream nuclear processes, such as ribosome assembly. To further corroborate this notion, this project is aimed at revealing nuclear co-import of a tetrameric complex composed of the biogenesis factors Enp1 and Ltv1, the ribosomal protein Rps3, and the Rps3-‘chaperone’ Yar1.4) Functional role of ubiquitin in the ubiquitin-fusion protein Rpl40This project should elucidate the effects on 60S subunit maturation due to impaired cleavage or the absence of the ubiquitin moiety from the natural ubiquitin-Rpl40 fusion protein Ubi1. Specifically, we aim to understand whether the ubiquitin moiety of Ubi1 serves principally as a cis-acting ‘chaperone’ for Rpl40 or whether it may fulfil additional roles during cytoplasmic pre-60S maturation events.