CE12 - Génétique, génomique et ARN

Initial events of large ribosomal subunit formation in eukaryotes – RIBOPRE60S

Submission summary

The ribosome catalyses peptide bond synthesis during the translation process. It is constituted by a large and a small subunit, each possessing an RNA scaffold bound by ribosomal proteins (RPs). The ribosome is a ribozyme since its catalytic center, carried by the large 60S ribosomal subunit, is made of ribosomal RNA (rRNA). Hence one of the key features of ribosome synthesis is the correct folding of the rRNAs to ensure optimum activity. Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles that contain pre-rRNAs, RPs, snoRNPs and assembly factors (AFs). The initial pre-rRNA folding and assembly events leading to the production of the first pre-60S particle, the first precursor to the 60S subunit, are of paramount importance for the formation of an active large ribosomal subunit. However, they feature among the least understood stages of ribosome biogenesis in eukaryotes. In the mature 60S subunit, the 25S and 5.8S rRNAs are organized in 6 structural domains (I to VI), each beginning with a root helix. The few available data concerning the first pre-60S particle indicate that root helix I and base-pairing interactions between 25S and 5.8S rRNAs, key determinants of domain I folding, are already established in this particle. It is envisaged that failure to correctly complete these and other unknown early folding events leads to the turnover of the first pre-60S particle. Several AFs contained within the first pre-60S particle, including the Npa1 complex composed of Npa1, Npa2, Nop8, Rsa3 and the Dbp6 DEAD-box protein, are required for its normal steady-state accumulation and termed “formation” (f)AFs. These fAFs could play crucial roles in early pre-rRNA folding events, but their function remains unknown. The first pre-60S particle also contains many snoRNPs involved in the methylation and pseudouridylation of specific 25S rRNA residues and whose associations with and dissociations from the pre-rRNA could impact its folding. We also produced evidence that one particular snoRNP containing the snoRNA snR190 could act as a dedicated pre-rRNA folding chaperone.
The aim of the RIBOPRE60S project is to decipher the molecular events leading to the formation of the first pre-60S particle in yeast. We will determine the function of fAFs and the snoRNP containing snR190 in the folding of the pre-rRNA constituent of the first pre-60S particle, in the assembly of RPs within this particle and in the control of snoRNA/pre-rRNA interactions. This requires to gain knowledge of where the fAFs bind on the pre-rRNA and which are their direct partners within this particle (RPs, AFs, or snoRNAs). It also requires to get insights into the structural organization of the first pre-60S particle, both in terms of pre-rRNA folding and global architecture. We will build on this information to determine how the absence or mutation of the fAFs impact the pre-rRNA folding and protein composition of the particle, as well as snoRNA/pre-rRNA interactions and pre-rRNA modifications. The project will also include a biochemical investigation of some key fAFs displaying enzymatic activities and will determine how their partners modulate these activities. We will focus in particular on Dbp6, the DEAD-box protein component of the Npa1 complex given the central role it plays in the early stages of large ribosomal subunit formation. Our work programme involves state of the art approaches to investigate protein/RNA interactions (in vivo CRAC), pre-rRNA folding (high throughput SHAPE), Npa1 complex and pre-60S particle structure (cryo-EM, CXMS), pre-ribosomal particle composition (label free quantitative mass spectrometry), in addition to standard biochemical assays to investigate enzymatic activities of DEAD-box proteins. We expect that this project will provide significant new insights into the earliest steps of large ribosomal subunit production in eukaryotes, in particular the molecular roles of fAFs in these steps.

Project coordination

Yves HENRY (Unité de biologie moléculaire, cellulaire et du développement)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CiTCoM Cibles Thérapeutiques et Conception de Médicaments
MCD Unité de biologie moléculaire, cellulaire et du développement

Help of the ANR 480,759 euros
Beginning and duration of the scientific project: March 2021 - 48 Months

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