Periodic Reporting for period 4 - Glowsome (Encapsulated eukaryotic ribosome assembly)
Reporting period: 2022-03-01 to 2022-08-31
1.1. Summary of the context and overall objectives of the project
The production of functional ribosomes is vital for every cell, with failure causing human diseases. Eukaryotic ribosome assembly is driven by ~200 assembly factors that guarantee efficient and accurate production of ribosomal subunits along a temporally and spatially ordered pathway. About one third of these factors are utilized in the formation of the earliest biogenesis intermediate, termed 90S pre-ribosome. Recent insight into the 90S structure from Chaetomium thermophilum and yeast has provided a first glimpse on a most sophisticated process, which includes RNA folding and processing, and the incorporation of ribosomal proteins. The nascent ribosomal RNA is co-transcriptionally mounted into a scaffold formed by numerous 90S factors. In this proposal we follow the idea that the 90S particle encapsulates the RNA transiently to protect it from unproductive interactions, allowing stepwise folding and maturation in a cascade of interdependent reactions and involvement of energy-consuming enzymes. The aim of this proposal is to decipher how these processes occur in an encapsulated environment, using Chaetomium thermophilum and yeast as models. The research will be based on techniques developed in my group to gain mechanistic insight into ribosome assembly, which could lead to a better understanding of how it is linked to other key cellular pathways and the development of diseases including cancer.
The objectives of this proposal are:
(i) Search for novel biogenesis intermediates that can retrace the 90S>pre-40S transition.
(ii) Development of in vitro assays to recapitulate steps in the transition from the 90S pre-ribosome to the pre-40S particle.
(iii) Analysis of 90S modules and their roles 90S pre-ribosome assembly and 90S to pre-40S maturation
(iv) Development of in vivo approaches in Chaetomium thermophilum to study the dynamic interaction of 90S assembly factors with pre-rRNA.
(v) In vivo generation of dominant-negative mutants of 90S factors to arrest ribosome biogenesis at distinct stages, with the goal of exploiting these intermediates for biochemical, structural and in vivo studies.
The production of functional ribosomes is vital for every cell, with failure causing human diseases. Eukaryotic ribosome assembly is driven by ~200 assembly factors that guarantee efficient and accurate production of ribosomal subunits along a temporally and spatially ordered pathway. About one third of these factors are utilized in the formation of the earliest biogenesis intermediate, termed 90S pre-ribosome. Recent insight into the 90S structure from Chaetomium thermophilum and yeast has provided a first glimpse on a most sophisticated process, which includes RNA folding and processing, and the incorporation of ribosomal proteins. The nascent ribosomal RNA is co-transcriptionally mounted into a scaffold formed by numerous 90S factors. In this proposal we follow the idea that the 90S particle encapsulates the RNA transiently to protect it from unproductive interactions, allowing stepwise folding and maturation in a cascade of interdependent reactions and involvement of energy-consuming enzymes. The aim of this proposal is to decipher how these processes occur in an encapsulated environment, using Chaetomium thermophilum and yeast as models. The research will be based on techniques developed in my group to gain mechanistic insight into ribosome assembly, which could lead to a better understanding of how it is linked to other key cellular pathways and the development of diseases including cancer.
The objectives of this proposal are:
(i) Search for novel biogenesis intermediates that can retrace the 90S>pre-40S transition.
(ii) Development of in vitro assays to recapitulate steps in the transition from the 90S pre-ribosome to the pre-40S particle.
(iii) Analysis of 90S modules and their roles 90S pre-ribosome assembly and 90S to pre-40S maturation
(iv) Development of in vivo approaches in Chaetomium thermophilum to study the dynamic interaction of 90S assembly factors with pre-rRNA.
(v) In vivo generation of dominant-negative mutants of 90S factors to arrest ribosome biogenesis at distinct stages, with the goal of exploiting these intermediates for biochemical, structural and in vivo studies.
1.2 Work performed from the beginning of the project (September 2017) to the end of the funding (August 2022) covered by the report and results achieved so far
For identification of regulatable promoters in Chaetomium thermophilum, we developed a minimal cultivation medium that allowed us to screen for xylose-inducible promoters in Chaetomium thermophilum. We have identified such promoters and validated them in vivo by using a thermo-stable YFP reporter. Hence, we cloned ca. 1.5 kb upstream (i.e. promoter) fragments of the xylose-regulated genes, derived from either the xylosidase-like (XYL) or xylitol dehydrogenase gene (XDH), fused them to the YFP reporter gene, and these constructs were transformed into C. thermophilum and tested for time-dependent YFP-expression upon glucose or xylose addition by fluorescence microscopy and Western blotting. Finally, we tested these xylose regulatable promoters for regulated expression of ribosome biogenesis factors followed by affinity-purification of pre-ribosomal particles. In particular interesting was a dominant negative ribosome biogenesis mutant rsa4 E114D. We could show that upon induced expression of ctRsa4 E114D using one of the xylose-regulatable promoters in Chaetomium thermophilium, the ribosomal protein reporter RpL25-YFP accumulated in the nucleus. All together this data showed that the identified regulatable promoters are suitable for induced expression studies in the thermophile. This in vivo work using the sugar-regulated thermophile promoters is also faciliatted by combining them with our recently developed homologous recombination system in Chaetomium thermophilum.
In the search for novel biogenesis intermediates that can retrace the 90S>pre-40S transition, we were able to describe for the first time how this 90S>pre-40S transition proceeds at a molecular level. Notably, the 90S pre-ribosomal particles have recruited the ATP-consuming Dhr1 helicase, which has triggered A1 pre-rRNA cleavage between 5’ ETS and 18S rRNA, and in a next step has dismantled the U3 snoRNP from the 90S yielding a so far completely unknown early pre-40S. In contrast to our current thinking that the 40S biogenesis pathway is simpler than the pre-60S assembly route, we discovered a series of novel maturation intermediates. (ii) On the basis of our new structural data, we can explain the coordinated pre-rRNA processing at sites A1 and A2. (iii) Our findings do not support the previous expectation in our field of a sudden release of the 5’-ETS particle and its associated major modules, but revealed the sequential and step-wise dismantling. These steps are reminiscent of a cascade-like reaction, where one step leads to the next one. (iv) Our cryo-EM ensemble revealed different conformations of the Dhr1 helicase in the various pre-ribosome states, suggesting how it functions in these successive steps.
During the course of studying the transition from the 90S pre-ribosome to the pre-40S particle, we discovered an unforeseen direct remodeling role of the nuclear RNA exosome. These insights were obtained based on biochemical and cryo-EM analyses, depicting the RNA exosome in a super-complex with the 90S pre-ribosome at a strategic position to initiate a number of irreversible dismantling and maturation steps. Thus, we could show that the exosome, rather than simply degrading the 5'-ETS RNA scaffold as previously assumed, is recruited to the 90S to initiate local 5'-ETS remodeling and degradation, crucial for the subsequent 90S to pre-40S transition.
We also could develop an ATP-dependent in vitro assay for the U3 release from Dis-C particles and followed this biochemically and by cryo-EM analysis. This revealed the ATP-dependent conversion of the primordial pre-40S, driven by the active Dhr1 helicase but not by an ATP-binding/hydrolysis defective Dhr1 mutant. The in vitro generated pre-40S particles, Dis-D and Dis-E, lost all the remaining 90S factors, but also the central pseudoknot has formed, a key tertiary RNA structure in the active mature 40S subunit. Thus, our data revealed that central pseudoknot formation is a spontaneous process towards the thermodynamic equilibrium, which can be initiated by U3 release. Moreover, during the Dhr1-driven U3 release, the 40S head started to adopt a more compact conformation, but it remained still very flexible, awaiting new pre-40S assembly factors to stabilize the head.
For identification of regulatable promoters in Chaetomium thermophilum, we developed a minimal cultivation medium that allowed us to screen for xylose-inducible promoters in Chaetomium thermophilum. We have identified such promoters and validated them in vivo by using a thermo-stable YFP reporter. Hence, we cloned ca. 1.5 kb upstream (i.e. promoter) fragments of the xylose-regulated genes, derived from either the xylosidase-like (XYL) or xylitol dehydrogenase gene (XDH), fused them to the YFP reporter gene, and these constructs were transformed into C. thermophilum and tested for time-dependent YFP-expression upon glucose or xylose addition by fluorescence microscopy and Western blotting. Finally, we tested these xylose regulatable promoters for regulated expression of ribosome biogenesis factors followed by affinity-purification of pre-ribosomal particles. In particular interesting was a dominant negative ribosome biogenesis mutant rsa4 E114D. We could show that upon induced expression of ctRsa4 E114D using one of the xylose-regulatable promoters in Chaetomium thermophilium, the ribosomal protein reporter RpL25-YFP accumulated in the nucleus. All together this data showed that the identified regulatable promoters are suitable for induced expression studies in the thermophile. This in vivo work using the sugar-regulated thermophile promoters is also faciliatted by combining them with our recently developed homologous recombination system in Chaetomium thermophilum.
In the search for novel biogenesis intermediates that can retrace the 90S>pre-40S transition, we were able to describe for the first time how this 90S>pre-40S transition proceeds at a molecular level. Notably, the 90S pre-ribosomal particles have recruited the ATP-consuming Dhr1 helicase, which has triggered A1 pre-rRNA cleavage between 5’ ETS and 18S rRNA, and in a next step has dismantled the U3 snoRNP from the 90S yielding a so far completely unknown early pre-40S. In contrast to our current thinking that the 40S biogenesis pathway is simpler than the pre-60S assembly route, we discovered a series of novel maturation intermediates. (ii) On the basis of our new structural data, we can explain the coordinated pre-rRNA processing at sites A1 and A2. (iii) Our findings do not support the previous expectation in our field of a sudden release of the 5’-ETS particle and its associated major modules, but revealed the sequential and step-wise dismantling. These steps are reminiscent of a cascade-like reaction, where one step leads to the next one. (iv) Our cryo-EM ensemble revealed different conformations of the Dhr1 helicase in the various pre-ribosome states, suggesting how it functions in these successive steps.
During the course of studying the transition from the 90S pre-ribosome to the pre-40S particle, we discovered an unforeseen direct remodeling role of the nuclear RNA exosome. These insights were obtained based on biochemical and cryo-EM analyses, depicting the RNA exosome in a super-complex with the 90S pre-ribosome at a strategic position to initiate a number of irreversible dismantling and maturation steps. Thus, we could show that the exosome, rather than simply degrading the 5'-ETS RNA scaffold as previously assumed, is recruited to the 90S to initiate local 5'-ETS remodeling and degradation, crucial for the subsequent 90S to pre-40S transition.
We also could develop an ATP-dependent in vitro assay for the U3 release from Dis-C particles and followed this biochemically and by cryo-EM analysis. This revealed the ATP-dependent conversion of the primordial pre-40S, driven by the active Dhr1 helicase but not by an ATP-binding/hydrolysis defective Dhr1 mutant. The in vitro generated pre-40S particles, Dis-D and Dis-E, lost all the remaining 90S factors, but also the central pseudoknot has formed, a key tertiary RNA structure in the active mature 40S subunit. Thus, our data revealed that central pseudoknot formation is a spontaneous process towards the thermodynamic equilibrium, which can be initiated by U3 release. Moreover, during the Dhr1-driven U3 release, the 40S head started to adopt a more compact conformation, but it remained still very flexible, awaiting new pre-40S assembly factors to stabilize the head.
1.3. Progress beyond the start of art and expected results at the end of the project
According to my ERC grant proposal formulated in 2017, all the suggested projects have been successfully conducted, yielding novel and conclusive data, of which most of them could be published in high profile peer-reviewed journals. Thus, I can conclude that the ERC grant could be effectively finished in August 2022, with groundbreaking insights into the earliest steps of eukaryotic ribosome assembly, specifically how the 90S pre-ribosome can develop into pre-40S particles to eventually mature into the ribosomal small subunit.
According to my ERC grant proposal formulated in 2017, all the suggested projects have been successfully conducted, yielding novel and conclusive data, of which most of them could be published in high profile peer-reviewed journals. Thus, I can conclude that the ERC grant could be effectively finished in August 2022, with groundbreaking insights into the earliest steps of eukaryotic ribosome assembly, specifically how the 90S pre-ribosome can develop into pre-40S particles to eventually mature into the ribosomal small subunit.