Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis.

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, reduces the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide-associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

Living cells are packed full of molecules known as proteins, which perform many vital tasks the cells need to survive and grow. Machines called ribosomes inside the cells use template molecules called messenger RNAs (or mRNAs for short) to produce proteins. The newly-made proteins then have to travel to a specific location in the cell to perform their tasks. Some newly-made proteins are prone to forming clumps, so cells have other proteins known as chaperones that ensure these clumps do not form. The ribosomes themselves are made up of several proteins, some of which are also prone to clumping as they are being produced. To prevent this from happening, cells control how many ribosomal proteins they make, so there are just enough to form the ribosomes the cell needs at any given time. Previous studies found that, in yeast, two ribosomal proteins called Rpl3 and Rpl4 each have their own dedicated chaperone to prevent them from clumping. However, it remained unclear whether these chaperones are also involved in regulating the levels of Rpl3 and Rpl4. To address this question, Pillet et al. studied both of these dedicated chaperones in yeast cells. The experiments showed that the chaperones bound to their target proteins (either units of Rpl3 or Rpl4) as they were being produced on the ribosomes. This protected the template mRNAs the ribosomes were using to produce these proteins from being destroyed, thus allowing further units of Rpl3 and Rpl4 to be produced. When enough Rpl3 and Rpl4 units were made, there were not enough of the chaperones to bind them all, leaving the mRNA templates unprotected. This led to the destruction of the mRNA templates, which decreased the numbers of Rpl3 and Rpl4 units being produced. The work of Pillet et al. reveals a feedback mechanism that allows yeast to tightly control the levels of Rpl3 and Rpl4. In the future, these findings may help us understand diseases caused by defects in ribosomal proteins, such as Diamond-Blackfan anemia, and possibly also neurodegenerative diseases caused by clumps of proteins forming in cells. The next step will be to find out whether the mechanism uncovered by Pillet et al. also exists in human and other mammalian cells.

Evolutionary and functional relationships in the ribosome biogenesis SBDS and EFL1 protein families.

Nascent ribosomal 60S subunits undergo the last maturation steps in the cytoplasm. The last one involves removing the anti-association factor eIF6 from the 60S ribosomal surface by the joint action of the Elongation Factor-like 1 (EFL1) GTPase and the SBDS protein. Herein, we studied the evolutionary relationship of the EFL1 and EF-2 protein families and the functional conservation within EFL1 orthologues. Phylogenetic analysis demonstrated that the EFL1 proteins are exclusive of eukaryotes and share an evolutionary origin with the EF-2 and EF-G protein families. EFL1 proteins originated by gene duplication from the EF-2 proteins and specialized in ribosome maturation while the latter retained their function in translation. Some organisms have more than one EFL1 protein resulting from alternative splicing, while others are encoded in different genes originated by gene duplication. However, the function of these alternative EFL1 proteins is still unknown. We performed GTPase activity and complementation assays to study the functional conservation of EFL1 homologs alone and together with their SBDS counterparts. None of the orthologues or cross-species combinations could replace the function of the corresponding yeast EFL1•SBDS binomial. The complementation of SBDS interspecies chimeras indicates that domain 2 is vital for its function together with EFL1 and the 60S subunit. The results suggest a functional species-specificity and possible co-evolution between EFL1, SBDS, and the 60S ribosomal subunit. These findings set the basis for further studies directed to understand the molecular evolution of these proteins and their impact on ribosome biogenesis and disease.

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae.

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

Interaction of the GTPase Elongation Factor Like-1 with the Shwachman-Diamond Syndrome Protein and Its Missense Mutations.

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2⁻3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.

Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in aShwachman-Diamond like syndrome.

BACKGROUND: For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome. OBJECTIVE: This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families. METHODS: Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants. RESULTS: Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1Δ yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells. CONCLUSIONS: Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.