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Journal of Biochemistry Jan 2022The level of ribosome biogenesis is highly associated with cell growth rate. Because many ribosomal proteins have extraribosomal functions, overexpression or...
The level of ribosome biogenesis is highly associated with cell growth rate. Because many ribosomal proteins have extraribosomal functions, overexpression or insufficient supply of these proteins may impair cellular growth. Therefore, the supply of ribosomal proteins is tightly controlled in response to rRNA syntheses and environmental stimuli. In our previous study, two RNA-binding proteins, Puf6 and Loc1, were identified as dedicated chaperones of the ribosomal protein eL43, with which they associate to maintain its protein level and proper loading. In this study, we demonstrate that Puf6 and Loc1 interact with RPL43 mRNA. Notably, Puf6 and Loc1 usually function as a dimeric complex to bind other mRNAs; however, in this instance, the individual proteins, but not the complex form, can bind RPL43 mRNA. Thus, Puf6 or Loc1 could bind RPL43 mRNA in loc1Δ or puf6Δ, respectively. The binding of Puf6 or Loc1 caused negative effects for eL43 production: decreased RNA stability and translation of RPL43A/B mRNA. The present results suggest that these dedicated chaperones control the protein levels of eL43 from the standpoint of stability and through regulating its production.
Topics: Nuclear Proteins; RNA, Messenger; RNA-Binding Proteins; Ribosomal Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34661244
DOI: 10.1093/jb/mvab110 -
PLoS Genetics Dec 2020The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is...
The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.
Topics: DEAD-box RNA Helicases; GTP-Binding Proteins; Methyltransferases; Mutation; Nuclear Proteins; Protein Binding; RNA, Small Nucleolar; RNA-Binding Proteins; Ribosomal Proteins; Ribosome Subunits, Small; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33306676
DOI: 10.1371/journal.pgen.1009215 -
Nature Mar 2020The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the...
The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.
Topics: Biocatalysis; Histones; Intrinsically Disordered Proteins; Microbial Viability; Nucleosomes; Phase Transition; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 32214243
DOI: 10.1038/s41586-020-2097-z -
Journal of Molecular Biology May 2018Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as...
Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.
Topics: Adaptor Proteins, Signal Transducing; Autophagy; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Cell Cycle Proteins; Gene Expression Regulation, Fungal; Mitosis; Nitrogen; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological
PubMed: 29694832
DOI: 10.1016/j.jmb.2018.04.007 -
The Journal of Biological Chemistry Nov 2021Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic...
Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.
Topics: Adaptor Proteins, Vesicular Transport; Cell Membrane; Cytoskeletal Proteins; Microfilament Proteins; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transport Vesicles; Vesicular Transport Proteins
PubMed: 34592316
DOI: 10.1016/j.jbc.2021.101254 -
Scientific Reports Sep 20166AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of...
6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.
Topics: Guanabenz; Heat-Shock Proteins; Mutation; Peptide Termination Factors; Phenanthridines; Prions; Protein Folding; RNA, Ribosomal; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 27633137
DOI: 10.1038/srep32117 -
Journal of Cellular Physiology Jun 2019"Moonlighting protein" is a term used to define a single protein with multiple functions and different activities that are not derived from gene fusions, multiple RNA... (Review)
Review
"Moonlighting protein" is a term used to define a single protein with multiple functions and different activities that are not derived from gene fusions, multiple RNA splicing, or the proteolytic activity of promiscuous enzymes. Different proteinous constituents of ribosomes have been shown to have important moonlighting extra-ribosomal functions. In this review, we introduce the impact of key moonlight ribosomal proteins and dependent signal transduction in the initiation and progression of various cancers. As a future perspective, the potential role of these moonlight ribosomal proteins in the diagnosis, prognosis, and development of novel strategies to improve the efficacy of therapies for human cancers has been suggested.
Topics: Carcinogenesis; Humans; Neoplasms; Protein Conformation; RNA, Ribosomal; Ribosomal Proteins; Ribosomes; Signal Transduction
PubMed: 30417503
DOI: 10.1002/jcp.27722 -
Journal of Cell Science Jul 2020PP2A (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase...
PP2A (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In , Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2A directly dephosphorylates Pds1 both and Pds1 hyperphosphorylation in a deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2A plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2A disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.
Topics: Cell Cycle Proteins; Chromosome Segregation; Nuclear Proteins; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Securin; Separase; Spindle Apparatus
PubMed: 32591482
DOI: 10.1242/jcs.243766 -
Nucleic Acids Research Apr 2022Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A...
Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.
Topics: Centromere; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Histones; Humans; Nucleocytoplasmic Transport Proteins; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 35234920
DOI: 10.1093/nar/gkac135 -
Journal of Molecular Biology Aug 2015In eukaryotes, nucleotide post-transcriptional modifications in RNAs play an essential role in cell proliferation by contributing to pre-ribosomal RNA processing,...
In eukaryotes, nucleotide post-transcriptional modifications in RNAs play an essential role in cell proliferation by contributing to pre-ribosomal RNA processing, ribosome assembly and activity. Box C/D small nucleolar ribonucleoparticles catalyze site-specific 2'-O-methylation of riboses, one of the most prevalent RNA modifications. They contain one guide RNA and four core proteins and their in vivo assembly requires numerous factors including (HUMAN/Yeast) BCD1/Bcd1p, NUFIP1/Rsa1p, ZNHIT3/Hit1p, the R2TP complex composed of protein PIH1D1/Pih1p and RPAP3/Tah1p that bridges the R2TP complex to the HSP90/Hsp82 chaperone and two AAA+ ATPases. We show that Tah1p can stabilize Pih1p in the absence of Hsp82 activity during the stationary phase of growth and consequently that the Tah1p:Pih1p interaction is sufficient for Pih1p stability. This prompted us to establish the solution structure of the Tah1p:Pih1p complex by NMR. The C-terminal tail S93-S111 of Tah1p snakes along Pih1p264-344 folded in a CS domain to form two intermolecular β-sheets and one covering loop. However, a thorough inspection of the NMR and crystal structures revealed structural differences that may be of functional importance. In addition, our NMR and isothermal titration calorimetry data revealed the formation of direct contacts between Pih1p257-344 and the Hsp82MC domain in the presence of Tah1p. By co-expression in Escherichia coli, we demonstrate that Pih1p has two other direct partners, the Rsa1p assembly factor and the Nop58p core protein, and in vivo and in vitro experiments mapped the required binding domains. Our data suggest that these two interactions are mutually exclusive. The implication of this finding for box C/D small nucleolar ribonucleoparticle assembly is discussed.
Topics: Escherichia coli; HSP90 Heat-Shock Proteins; Hydrophobic and Hydrophilic Interactions; Methylation; Molecular Chaperones; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Protein Binding; Protein Folding; Protein Interaction Maps; Protein Structure, Tertiary; RNA, Ribosomal; Ribonucleoproteins, Small Nuclear; Ribonucleoproteins, Small Nucleolar; Ribosomal Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26210662
DOI: 10.1016/j.jmb.2015.07.012