Project description:Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes – UtpA and UtpB – interact with nascent pre-ribosomal RNA have so far been poorly understood. We have combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking to elucidate the essential function of both complexes. Here we show that UtpA contains a large composite RNA binding site and captures the 5´ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to key architectural sites such as an RNA duplex formed by the 5´ ETS and U3 snoRNA as well as the 3´ boundary of 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.
Project description:Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The ‘U three protein’ (UTP) complexes and snoRNA particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5’-end of the pre-ribosomal RNA (5’-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5’-ETS RNA from an internally hidden 5’-end up to the region that hybridizes to the 3’-hinge sequence of U3 snoRNA and validate a specific Utp4/5’-ETS interaction by CRAC analysis. Altogether Utp4 is central to the UTP-A complex and organizes the 5’-ETS for further maturation.
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. CLIP-seq was performed in Flag-SIRT7-293T cells.
Project description:Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we find that the universal cotranslational machinery adapts to accommodate the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). During eEF1A synthesis, chaperone Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult to fold protein that necessitates dedicated folding factor Chp1 at the ribosomal tunnel exit to protect the eukaryotic cell from proteostasis collapse.
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA.
Project description:The ribosome associated complex (RAC) is a ribosome bound protein chaperone complex reported to surveil the translation of proteins with positively charged regions. It has been posited that RAC might be able to directly regulate translation by coupling co-translational folding with the peptide-elongation cycle. To identify the targets of RAC in cells and test the hypothesis that the complex modulates translation, we performed ribosome profiling on wild- type yeast cells and cells lacking a key component of the RAC that binds near the ribosome active site (zuo1Δ). Ribosome profiling is a sequencing-based technique that allows us to take a nucleotide resolution snapshot of where every ribosome sits on every mRNA in a cell at a given point in time. This powerful approach can provide information about the contributions of individual proteins to the translational landscape of a cell. We identified >300 targets for the RAC, and unexpectedly observed that the ribosome frameshifts on ~10% of these targets in zuo1Δ cells. The maintenance of ribosome reading frame is essential for cell health because frameshifts can result in the production of non-functional truncated and extended protein products. These studies have the potential to uncover RAC as a critical determinant of translational fidelity in eukaryotic cells.
Project description:Ribosome biosynthesis plays a crucial role in regulating protein translation and is essential for cell growth and development in physiological progress. The progression and recurrence of Pterygia mainly occur due to the abnormal proliferation and migration of stromal Pterygia fibroblasts. Small nucleolar RNA U3 (U3 snoRNA), harboring the atypical C/D boxes, is involved in 18S ribosomal RNA (18S rRNA) synthesis; however, the mechanism of U3 snoRNA in Pterygia remains unclear. Methods: Primary HCFs and HPFs were separated and cultured from fresh conjunctival grafts and Pterygia tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPF and HCF cells for further specific phenotypes analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in Pterygia promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5′ external transcribed spacer (5′ ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusion: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in Pterygia through modulating the 18S RNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of Pterygia.
Project description:Proteostasis is a fundamental network of cellular pathways that ensures the optimal concentration and composition of correctly folded proteins within cells in normal and stress conditions. Among key components of this network are the molecular chaperones, which mediate protein folding but also act as modulators of protein synthesis. We have reported on a functional link between translation and de novo folding of proteins in the yeast Saccharomyces cerevisiae by uncovering a specific synthetic-lethal interaction between apparent unrelated mutant variants, the uL3[W255C] variant of the ribosomal protein uL3 and the null mutants of Zuo1 and Ssz1. Zuo1 and Ssz1 are components of the chaperone system named as ribosome-associated complex. Here, we performed a genome-wide analysis of ribosome dynamics by 5PSeq (Pelechano et al. 2015 PMID 2604644) in strains harbouring either wild-type uL3 or mutant uL3[W255C] in the presence or absence of Zuo1 or Ssz1. This method allows the study of ribosome dynamics, by sequencing 5’ phosphorylated mRNA co-translational degradation intermediates. Our results indicate that the rpl3[W255C] mutant is slightly impaired in translation elongation, defect that is significantly enhanced when combined with the deprivation of either Zuo1 or Ssz1.