ABSTRACT
In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.
Subject(s)
Cell Cycle Proteins , Heterochromatin , Histone-Lysine N-Methyltransferase , Histones , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Cell Cycle Proteins/metabolism , Centromere/metabolism , Heterochromatin/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Methylation , Methyltransferases/metabolism , RNA, Long Noncoding/metabolism , RNA, Long Noncoding/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism , RNA, Fungal/genetics , RNA, Small Interfering/geneticsABSTRACT
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Subject(s)
Eukaryotic Initiation Factor-4F , Eukaryotic Initiation Factor-4G , Heat-Shock Response , Poly(A)-Binding Proteins , Protein Biosynthesis , RNA, Messenger , Ribonucleoproteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Heat-Shock Response/genetics , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4F/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Eukaryotic Initiation Factor-4G/genetics , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4A/genetics , Gene Expression Regulation, Fungal , Protein Binding , RNA, Fungal/metabolism , RNA, Fungal/geneticsABSTRACT
Heterochromatin is a key architectural feature of eukaryotic chromosomes, which endows particular genomic domains with specific functional properties. The capacity of heterochromatin to restrain the activity of mobile elements, isolate DNA repair in repetitive regions and ensure accurate chromosome segregation is crucial for maintaining genomic stability. Nucleosomes at heterochromatin regions display histone post-translational modifications that contribute to developmental regulation by restricting lineage-specific gene expression. The mechanisms of heterochromatin establishment and of heterochromatin maintenance are separable and involve the ability of sequence-specific factors bound to nascent transcripts to recruit chromatin-modifying enzymes. Heterochromatin can spread along the chromatin from nucleation sites. The propensity of heterochromatin to promote its own spreading and inheritance is counteracted by inhibitory factors. Because of its importance for chromosome function, heterochromatin has key roles in the pathogenesis of various human diseases. In this Review, we discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin Assembly and Disassembly/physiology , Heterochromatin/genetics , Heterochromatin/metabolism , Aging, Premature/genetics , Animals , Cell Differentiation/genetics , DNA Methylation , DNA Repair , Epigenesis, Genetic , Gene Silencing , Humans , Models, Biological , Obesity/genetics , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Virus Latency/geneticsABSTRACT
mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.
Subject(s)
Codon , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Protein Biosynthesis , RNA Stability , RNA, Fungal/chemistry , RNA, Messenger/chemistryABSTRACT
Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.
Subject(s)
Cryoelectron Microscopy , Exoribonucleases , RNA Polymerase II , RNA, Messenger , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Transcription Termination, Genetic , Exoribonucleases/chemistry , Exoribonucleases/metabolism , Exoribonucleases/ultrastructure , Models, Molecular , Protein Binding , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Polymerase II/ultrastructure , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/ultrastructure , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/ultrastructure , Protein Domains , RNA, Fungal/biosynthesis , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/ultrastructureABSTRACT
The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.
Subject(s)
Chromosome Segregation , Escherichia coli/genetics , Meiosis , RNA Processing, Post-Transcriptional , RNA, Bacterial/genetics , RNA, Fungal/genetics , RNA, Messenger/genetics , Schizosaccharomyces/genetics , Uridine/metabolism , Chromosomes, Bacterial , Chromosomes, Fungal , Chromosomes, Human , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Evolution, Molecular , HCT116 Cells , Humans , Oxidation-Reduction , RNA, Bacterial/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/metabolism , Sequence Analysis, RNA , Tubulin/genetics , Tubulin/metabolismABSTRACT
In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.
Subject(s)
Chromatin Assembly Factor-1/metabolism , Chromatin/metabolism , Gene Expression Regulation, Fungal , RNA, Fungal/genetics , RNA, Untranslated/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Chromatin Assembly and Disassembly , Nucleosomes/metabolism , Promoter Regions, Genetic , RNA Stability , RNA, Fungal/metabolism , RNA, Untranslated/metabolism , Transcription Termination, Genetic , Transcription, GeneticABSTRACT
The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.
Subject(s)
Models, Chemical , RNA Splicing , RNA, Fungal/chemistry , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Spliceosomes/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/metabolismABSTRACT
Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 3' regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription.
Subject(s)
RNA, Fungal/genetics , RNA, Untranslated/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Termination, Genetic , Transcriptome , Down-Regulation , Nuclear Proteins/metabolism , Promoter Regions, Genetic , RNA, Antisense/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Maintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin-preferentially â¼30 bp upstream of transcription start-sites-and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.
Subject(s)
Gene Expression Regulation, Fungal , RNA Stability , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , Cell Nucleus/metabolism , Cytoplasm/metabolism , Exoribonucleases/metabolism , Genes, Fungal/genetics , RNA Polymerase II/metabolism , RNA, Fungal/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
Subject(s)
RNA Polymerase III/genetics , RNA Polymerase II/genetics , RNA Polymerase I/genetics , RNA, Fungal/chemistry , Saccharomyces cerevisiae/genetics , Transcription Elongation, Genetic , Base Composition , Base Sequence , Binding Sites , Chromatin/chemistry , Chromatin/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Gene Expression Regulation, Fungal , Protein Binding , RNA Folding , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , RNA Polymerase III/metabolism , RNA Splice Sites , RNA Splicing , RNA, Fungal/genetics , RNA, Fungal/metabolism , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , ThermodynamicsABSTRACT
The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.
Subject(s)
RNA, Ribosomal, 18S , Ribosomes , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Ribosomes/metabolism , RNA Cleavage , RNA Stability/genetics , RNA, Fungal/metabolism , RNA, Fungal/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Ribosomal, 18S/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.
Subject(s)
Gene Expression Regulation, Fungal , Genes, Mating Type, Fungal , RNA, Fungal/genetics , RNA, Long Noncoding/genetics , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Gametogenesis , Nuclear Proteins/genetics , Promoter Regions, Genetic , Repressor Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spores, Fungal , Transcription Factors/geneticsABSTRACT
The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.
Subject(s)
Gene Expression Regulation, Fungal , Histone Deacetylases/metabolism , RNA, Antisense/genetics , RNA, Fungal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , Histones/metabolism , Kinetics , Methylation , Promoter Regions, Genetic , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & developmentABSTRACT
Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.
Subject(s)
Chaetomium/metabolism , Fungal Proteins/metabolism , Nucleic Acid Conformation , RNA, Fungal/metabolism , RNA, Ribosomal, 18S/metabolism , Ribosomes/metabolism , Chaetomium/genetics , Fungal Proteins/genetics , RNA, Fungal/genetics , RNA, Ribosomal, 18S/genetics , Ribosomes/geneticsABSTRACT
Ribosome is the most abundant RNA-protein complex in a cell, and many copies of the ribosomal RNA gene (rDNA) have to be maintained. However, arrays of tandemly repeated rDNA genes can lose the copies by intra-repeat recombination. Loss of the rDNA copies of Saccharomyces cerevisiae is counteracted by gene amplification whereby the number of rDNA repeats stabilizes around 150 copies, suggesting the presence of a monitoring mechanism that counts and adjusts the number. Here, we report that, in response to rDNA copy loss, the upstream activating factor (UAF) for RNA polymerase I that transcribes the rDNA is released and directly binds to a RNA polymerase II-transcribed gene, SIR2, whose gene products silence rDNA recombination, to repress. We show that the amount of UAF determines the rDNA copy number that is stably maintained. UAF ensures rDNA production not only by rDNA transcription activation but also by its copy-number maintenance.
Subject(s)
DNA Copy Number Variations , Gene Dosage , RNA Polymerase I/metabolism , RNA, Fungal/genetics , RNA, Ribosomal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Binding Sites , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Protein Binding , RNA Polymerase I/genetics , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/genetics , Sirtuin 2/metabolism , Transcription Factors/geneticsABSTRACT
Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked from or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene subclasses, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.
Subject(s)
Gene Expression Regulation, Fungal , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcriptome , Cell Proliferation , Mutation , Protein Processing, Post-Translational , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Time FactorsABSTRACT
Macroautophagy/autophagy is a key catabolic recycling pathway that requires fine-tuned regulation to prevent pathologies and preserve homeostasis. Here, we report a new post-transcriptional pathway regulating autophagy involving the Pat1-Lsm (Lsm1 to Lsm7) mRNA-binding complex. Under nitrogen-starvation conditions, Pat1-Lsm binds a specific subset of autophagy-related (ATG) transcripts and prevents their 3' to 5' degradation by the exosome complex, leading to ATG mRNA stabilization and accumulation. This process is regulated through Pat1 dephosphorylation, is necessary for the efficient expression of specific Atg proteins, and is required for robust autophagy induction during nitrogen starvation. To the best of our knowledge, this work presents the first example of ATG transcript regulation via 3' binding factors and exosomal degradation.
Subject(s)
Autophagy-Related Proteins/metabolism , Autophagy , Nitrogen/deficiency , RNA Cap-Binding Proteins/metabolism , RNA Stability , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , 3' Untranslated Regions , Autophagy-Related Proteins/genetics , Binding Sites , Gene Expression Regulation, Fungal , Humans , Jurkat Cells , Multiprotein Complexes , Phosphorylation , Protein Binding , RNA Cap-Binding Proteins/genetics , RNA, Fungal/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal TransductionABSTRACT
Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
Subject(s)
Endonucleases/metabolism , Polyadenylation , RNA Precursors/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , mRNA Cleavage and Polyadenylation Factors/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Cytochromes c/genetics , Cytochromes c/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases/genetics , Enzyme Activation , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Docking Simulation , Multiprotein Complexes , Polynucleotide Adenylyltransferase/genetics , Polynucleotide Adenylyltransferase/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA Precursors/genetics , RNA, Fungal/genetics , RNA, Messenger/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Tandem Mass Spectrometry , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.