chaperone; Ribosome biogenesis; Saccharomyces cerevisiae; AAA-ATPase; yeast genetics
Thoms Matthias, Mitterer Valentin, Kater Lukas, Falquet Laurent, Beckmann Roland, Kressler Dieter, Hurt Ed (2018), Suppressor mutations in Rpf2–Rrs1 or Rpl5 bypass the Cgr1 function for pre-ribosomal 5S RNP-rotation, in Nature Communications
, 9(1), 4094-4094.
Kater Lukas, Thoms Matthias, Barrio-Garcia Clara, Cheng Jingdong, Ismail Sherif, Ahmed Yasar Luqman, Bange Gert, Kressler Dieter, Berninghausen Otto, Sinning Irmgard, Hurt Ed, Beckmann Roland (2017), Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes, in Cell
, 171(7), 1599-1610.e14.
Kressler Dieter, Hurt Ed, Baßler Jochen (2017), A Puzzle of Life: Crafting Ribosomal Subunits, in Trends in Biochemical Sciences
, 42(8), 640-654.
Pillet Benjamin, Mitterer Valentin, Kressler Dieter, Pertschy Brigitte (2017), Hold on to your friends: Dedicated chaperones of ribosomal proteins, in BioEssays
, 39(1), e201600153-e201600153.
Mitterer Valentin, Gantenbein Nadine, Birner-Gruenberger Ruth, Murat Guillaume, Bergler Helmut, Kressler Dieter, Pertschy Brigitte (2016), Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1, in SCIENTIFIC REPORTS
, 6, 36714.
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.
Mitterer Valentin, Murat Guillaume, Réty Stéphane, 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, 10336-10336.
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, 7494-7494.
Rodríguez-Galán Olga, García-Gómez Juan J, Kressler Dieter, de la Cruz Jesús (2015), Immature large ribosomal subunits containing the 7S pre-rRNA can engage in translation in Saccharomyces cerevisiae., in RNA biology
, 12(8), 838-46.
Fernández-Pevida Antonio, Kressler Dieter, de la Cruz Jesús (2015), Processing of preribosomal RNA in Saccharomyces cerevisiae., in Wiley interdisciplinary reviews. RNA
, 6(2), 191-209.
Calviño Fabiola R, Kharde Satyavati, Ori Alessandro, Hendricks Astrid, Wild Klemens, Kressler Dieter, Bange Gert, Hurt Ed, Beck Martin, Sinning Irmgard (2015), Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site., in Nature communications
, 6, 6510-6510.
Pillet Benjamin, García-Gómez Juan J, Pausch Patrick, Falquet Laurent, Bange Gert, de la Cruz Jesús, Kressler Dieter (2015), The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site., in PLoS genetics
, 11(10), 1005565-1005565.
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. 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. In order to understand the process at a molecular level, it will be necessary to gain structural insight into the trans-acting factors and the pre-ribosomal particles that they are associated with and acting on. Finally, we will have to understand how ribosomal proteins contribute to ribosome biogenesis and how ribosomal proteins and trans-acting factors get 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. Here, we will exploit the recently solved Nsa1 crystal structure in order to identify by mutational analysis the Nsa1 surface 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) Investigation of assembly alterations that bypass the requirement for pre-60S association of Nsa1Our recent studies revealed that the requirement for pre-60S association of the essential Nsa1 is bypassed by reduced functionality of several 60S biogenesis factors (Ebp2, Mak5, Nop1, and Nop4). In order to obtain a comprehensive inventory of assembly alterations that render Nsa1 binding dispensable, we will further exploit this ’in vivo structure probing’ approach by isolating and characterizing novel ?nsa1 suppressors. Alterations of local (pre-)rRNP structures within these pre-60S particles will then be experimentally determined by chemical probing.3) Identification of additional nuclear and/or pre-60S substrates of the AAA-type ATPase Rix7Our observation that dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1 strongly suggests that Rix7 may have additional nuclear substrates besides Nsa1. Moreover, over-expression of wild-type Rix7 has a negative effect on growth of mak5 and ebp2 mutant cells, both in the absence and presence of Nsa1, indicating that Rix7 may act on structurally defective pre-60S subunits and subject these to degradation. To further explore these exciting possibilities, the power of yeast genetics will be exploited to identify additional nuclear substrates of Rix7 and to unveil how Rix7 recognizes structurally defective pre-60S subunits.4) 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 already revealed for a set of four ribosomal proteins (Rps3, Rpl3, Rpl5, and Rpl10) that the ‘chaperone’ partners (Yar1, Rrb1, Syo1, and Sqt1) recognize their very N-terminal residues, notably implicated in rRNA binding. 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 and functional analyses of the ‘chaperone’/ribosomal protein interactions.5) 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.