Ribosome biogenesis; AAA-ATPases; Saccharomyces cerevisiae; ubiquitin; pre-ribosomal particles; TAP purification; genetics; ribosomal proteins; molecular biology; cell biology; yeast genetics; chaperones
Garcia-Gomez Juan J., Fernandez-Pevida Antonio, Lebaron Simon, Rosado Ivan V., Tollervey David, Kressler Dieter, de la Cruz Jesus (2014), Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3, in PLOS GENETICS
, 10(3), e1004205.
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.
Bange Gert, Murat Guillaume, Sinning Irmgard, Hurt Ed, Kressler Dieter (2013), New twist to nuclear import: When two travel together., in Communicative & integrative biology
, 6(4), 24792-24792.
Kressler Dieter, Bange Gert, Ogawa Yutaka, Stjepanovic Goran, Bradatsch Bettina, Pratte Dagmar, Amlacher Stefan, Strauß Daniela, Yoneda Yoshihiro, Katahira Jun, Sinning Irmgard, Hurt Ed (2012), Synchronizing nuclear import of ribosomal proteins with ribosome assembly, in Science
, 338, 666-671.
Kressler Dieter, Hurt Ed, Bergler Helmut, Bassler Jochen (2012), The power of AAA-ATPases on the road of pre-60S ribosome maturation--molecular machines that strip pre-ribosomal particles., in Biochimica et biophysica acta
, 1823(1), 92-100.
Koch Barbara, Mitterer Valentin, Niederhauser Johannes, Stanborough Tamsyn, Murat Guillaume, Rechberger Gerald, Bergler Helmut, Kressler Dieter, Pertschy Brigitte (2012), Yar1 protects the ribosomal protein rps3 from aggregation., in The Journal of biological chemistry
, 287(26), 21806-15.
Fernandez-Pevida Antonio, Rodriguez-Galan Olga, Diaz-Quintana Antonio, Kressler Dieter, de la Cruz Jesus (2012), Yeast ribosomal protein L40 assembles late into pre-60S ribosomes and is required for their cytoplasmic maturation, in Journal of Biological Chemistry
, 287, 38390-38407.
Bange Gert, Kümmerer Nico, Grudnik Przemyslaw, Lindner Robert, Petzold Georg, Kressler Dieter, Hurt Ed, Wild Klemens, Sinning Irmgard (2011), Structural basis for the molecular evolution of SRP-GTPase activation by protein., in Nature structural & molecular biology
, 18(12), 1376-80.
Kressler Dieter, Hurt Ed, Bassler Jochen (2010), Driving ribosome assembly., in Biochimica et biophysica acta
, 1803(6), 673-83.
The process of ribosome biogenesis is evolutionarily conserved amongst 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 (>150) 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 formation are still largely unknown. 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. In addition, the contribution of ribosomal proteins to the biogenesis of ribosomal subunits has to be further explored. 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) Mechanistic dissection of the Rix7-mediated release of Nsa1 from pre-60S particlesWe have shown that the AAA-type ATPase Rix7 is required for the release of Nsa1 from a discrete, late nucleolar pre-60S ribosomal particle, thereby triggering the progression of 60S biogenesis. Furthermore, we have recently solved, in collaboration with the group of I. Sinning (Universität Heidelberg), the crystal structure of Nsa1, which forms a 7-bladed WD-repeat ß-propeller. Additionally, our unpublished results reveal that the first predicted a-helix (termed D2-H1) of Rix7’s D2 AAA-domain contributes to Nsa1 binding and that Nsa1 is polyubiquitinated in vivo. Therefore, one objective of my research proposal is to understand the molecular details of how Rix7 mediates the release of Nsa1 from pre-60S particles. Specifically, we wish to determine how Rix7 recognizes Nsa1 on the pre-ribosomal particle and how ubiquitination of Nsa1 contributes to its release. In a sub-project, we will assess the functional significance of D2-H1 in Cdc48, the closest yeast homologue of Rix7, thereby possibly deducing new mechanistic insights of type II AAA-ATPase function.2) Analysis of the Nsa1/pre-ribosome interaction at a structural levelWe 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 wish to determine the binding site of Nsa1 on pre-60S ribosomes. This study should allow us to gain insight into the possible structural rearrangements within pre-60S particles that will be promoted by the release of Nsa1.3) Regulation of ribosome biogenesis by ubiquitinationThe contribution of post-translational modifications to the regulation of ribosome biogenesis has so far only been scarcely investigated. However, recent evidence suggests that sumoylation and ubiquitination may have an impact on ribosome formation. To address how ubiquitination may regulate individual steps of ribosome assembly and/or target aberrant pre-ribosomal particles for degradation, we will determine in a global approach which protein trans-acting factors are modified by ubiquitin. Specifically, we wish to (i) determine whether the observed modification serves a regulatory purpose or targets for proteasomal degradation, (ii) understand its functional importance, and (iii) identify the E2 and E3 enzymes that mediate ubiquitination.4) Contribution of ribosomal proteins and their ’chaperones’ to ribosome formationThis project is aimed at the identification and functional characterization of ‘chaperones’ of ribosomal proteins and should therefore shed light on how ribosomal proteins are stably expressed, delivered to their site of assembly, and efficiently assembled into ribosomal subunits. In validation of this approach, we have identified the previously uncharacterized protein Ydl063c, for which we could already show an involvement in ribosome biogenesis, as a binding partner of Rpl5. With this study, we will further explore the interplay between Rpl5 and Ydl063c at a functional and structural level. In a sub-project, we will, in collaboration with the group of J. de la Cruz (Universidad de Sevilla), explore the intriguing role of the large subunit ribosomal protein Rpl3 in 40S subunit biogenesis and assess whether processing of the 20S pre-rRNA occurs on translating ribosomes.