Understanding assembly of macromolecular complexes using C/D snoRNPs as a model – snoRNPASSEMBLY

Using both the human and yeast system, we will follow five axes: (i) proteomic approaches to characterize the assembly process in vivo and to identify small building blocks in the assembly reaction; (ii) systematic interactions studies to map protein-protein and protein-RNA interaction sites at the level of single amino-acids and nucleotides; (iii) structural studies of key sub-complexes (NMR, X-ray, structural mass-spectrometry, cryo-EM) and use of integrative strategies for larger assembly intermediates; (iv) development of an in vitro assembly system amenable to biochemical/biophysical experiments; (v) functional experiments in vivo or in vitro.

*Our consortium has discovered the existence of a new R2TP-like co-chaperone, called R2SP (Maurizy et al., Nat Commun 2018). It comprises the proteins RUVBL1/2, PIH1D2 (homolog to PIH1D1), and SPAG1 (homolog to RPAP3). R2SP also function in quaternary protein folding, like R2TP.

*We have completed a detailed structural analysis of the domains of RPAP3, alone and in complex with fragments of HSP70, HSP90 and PIH1D1. This has been published in Henri et al., Structure 2018.

*Our consortium has discovered the existence of a new R2TP-like co-chaperone, called R2SP. It comprises the proteins RUVBL1/2, PIH1D2 (homolog to PIH1D1), and SPAG1 (homolog to RPAP3). R2SP also function in quaternary protein folding, like R2TP.

*We have completed a detailed structural analysis of the domains of RPAP3, alone and in complex with fragments of HSP70, HSP90 and PIH1D1.

1-Henri J., Chagot M-E., Bourguet M., Abel Y., Terral G., Maurizy C., Aigueperse C., Georgescauld F., Vandermoere F., Saint-Fort R., Behm-Ansmant I., Charpentier B., Pradet-Balade B., Verheggen C., Bertand E., Meyer P., Cianférani S., Manival X., Quinternet M. Deep structural analysis of RPAP3 and PIH1D1, two components of the HPS90 co-chaperone R2TP complex, Structure, 2018, 26, 1196-1209.

2-Maurizy C., Quinternet M., Abel Y., Verheggen C.,. Santo P E, Bourguet M., Paiva A.C.F., Bragantini B., Chagot M-E., Robert M-C., Abeza C., Fabre P., Fort P., Vandermoere F, Sousa P.M. F., Rain J-C., Charpentier B., Bandeiras T-M., Cianférani S., Pradet-Balade B., Manival X., Bertrand E. The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones. Nature Communications, 2018, 9, 2093

Many cellular functions are performed by molecular machines made of RNA-protein complexes. This includes the ribosome and spliceosome, and also numerous other stable RNP particles involved in various aspects of cellular life. For the cell, it is essential to produce these RNP particles in a functional state and this can be a real challenge. Studies performed on many systems have shown that assembling RNP particles requires a large number of dedicated factors. These factors can chaperone free subunits, increase the specificity of assembly, transport subunits, and control the quality of the particle.

Given the importance of assembly machineries for RNP particles, it is not surprising that they also occur for stable protein complexes that do not contain RNAs. Recent studies showed that assembly factors for protein complexes are linked to the protein folding machinery. Indeed, quaternary folding can be seen as an extension of tertiary folding, with the difference that it occurs in trans and not in cis. This however introduces specific difficulties with regard to stoichiometry and concentration of free subunits. Because many molecular machines are made of stable multi-subunit assemblies, understanding how they are built is an important question in cell biology.

HSP90 is a highly conserved chaperone that is involved in the late stages of protein folding. It forms a dimeric molecular clamp whose conformation is driven by ATP binding and hydrolysis, and HSP90 co-chaperones regulates its ATPase cycle. Recently, the cochaperone R2TP was shown to be involved in the assembly of macromolecular complexes. Indeed, the HSP90/R2TP system has been shown to be involved in the assembly of key cellular machineries: RNA polymerases, snoRNPs, snRNPs, telomerase, and complexes containing any of the PIKKs (mTOR, ATM/ATR, DNA-PK, TRRAP and SMG1).

Our aim is to decipher the mechanisms of action of the HSP90/R2TP system, using box C/D snoRNP as a model. We want to provide innovative ideas applicable to other assembly machineries.

Our previous results suggest a three step mechanism for the assembly of box C/D snoRNP: (i) one client would be loaded on HSP90 and stabilized by the chaperone; (ii) a second client would be loaded on HSP90 by the R2TP, and the chaperone would promote association between the two clients; (iii) the R2TP would tranfer the associated clients to the RUVBL1/2 AAA+ ATPases. The resulting complex containing RUVBL1/2 and the assembled clients would be stable and could proceed for further assembly steps. Upon completion of the process, the RUVBL1/2 would recycle back to the R2TP. This model solves the problem of unstable assembly intermediates. It also proposes that the chaperone directly participates in the assembly process and further clarifies the molecular function of the RUVBL1/2 proteins, which remained so far elusive.

Using both the human and yeast system, we will follow five axes: (i) proteomic approaches to characterize the assembly process in vivo and to identify small building blocks in the assembly reaction; (ii) systematic interactions studies to map protein-protein and protein-RNA interaction sites at the level of single amino-acids and nucleotides (mutagenesis screens, CRAC); (iii) structural studies of key sub-complexes (NMR, X-ray, structural mass-spectrometry, cryo-EM) and use of integrative strategies for larger assembly intermediates; (iv) development of an in vitro assembly system amenable to biochemical/biophysical experiments; (v) functional experiments in vivo or in vitro.

Our consortium has the requested expertise and also many preliminary results to maximize success. Given the variety of the known R2TP clients, as well as the involvement of the RUVBL1/2 ATPases in many cellular processes, this project will have a direct impact in many different fields. In addition, our project will generate insights into quaternary protein folding, a very basic and fundamental question that is still poorly understood.