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During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus. Here, using cryo-EM, the authors detail how the yeast co-transcriptional assembly of the large ribosomal subunit involves a quality-control checkpoint. Ribosome-assembly factors implement this checkpoint by probing the formation and ensuring the co-operative stabilization of correctly folded ribosomal RNA.

Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD+-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT (‘facilitates chromatin transcription’) in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.

Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

Cdc42, a small GTPase essential for cell polarity, often becomes hyperactive with age and promotes senescence in yeast and animal cells. Yet, the mechanisms driving its age-related upregulation remain unclear. Here, we show that in budding yeast, Cdc42 accumulates over successive cell divisions and that reducing its levels extends life span. Using microfluidics-assisted live-cell imaging and genetic analysis, we found that Cdc42 is distributed unevenly between mother and daughter cells during division. Daughter cells inherit lower Cdc42 levels, which likely help them remain young. This asymmetric distribution depends on Cdc42’s association with and/or release from endomembranes and likely involves Ydj1, a farnesylated Hsp40/DnaJ chaperone anchored to the endoplasmic reticulum. Ydj1 interacts with Cdc42, promoting its stability and proper partitioning during cell division. We propose that ER-bound Ydj1 facilitates the asymmetric distribution of Cdc42, thereby restricting aging to mother cells.

Cohesin mediates central features of chromosome architecture. The protein complex governs sister chromatid cohesion and organizes genomes into loops and domains. In budding yeast, cohesin accumulates at discrete sites on chromosome arms, as well as at domains of heterochromatin. The molecular basis for the distribution at these sites is not yet resolved, although the heterochromatin protein Sir2 has been implicated. If cohesin were to bind a recurring feature of heterochromatin, then size of the cohesin domain would match the size of the heterochromatin domain. To test this hypothesis, the span of heterochromatin at the HMR silent mating-type locus was truncated with artificial barrier elements built from bacterial DNA binding proteins. We found that the most effective barrier reduced the footprint of Sir3 and Smc3, representative components of yeast heterochromatin and cohesin. These results show that reducing the span of heterochromatin at HMR constrains the size of the cohesin bound domain.

Gene silencing at the mating-type loci in budding yeast ( HMRa and HMLα ) depends on the Sir proteins. Sir2, Sir3 and Sir4 are indispensable, whereas Sir1 has a limited role. Sir proteins are also involved in repression at telomeres and rDNA repeats. Sir proteins may mediate silencing by limiting access to DNA. Using an inducible DNA methyltransferase expression system, we showed previously that the silenced mating-type loci are methylated at a much slower rate than the rest of the genome in vivo. Here, we show that Sir2, Sir3 and Sir4 are all required to impede access to the mating-type loci and telomeric X-elements. rDNA access is impeded by Sir2 and Sir3, but not Sir4. Methylation rates at adjacent rDNA repeats are not strongly correlated, suggesting that Sir proteins silence rDNA repeats randomly. Sir1 is required to impede access to HMRa , but not to HMLα , telomeres or rDNA. Since silenced DNA is accessible in vivo, albeit at a slower rate than elsewhere in the genome, steric occlusion is unlikely to be the primary mechanism of silencing.

In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility in old cells. This process is thought to reflect a decrease in the activity of the NAD⁺ (oxidized nicotinamide adenine dinucleotide)–dependent deacetylase Sir2. We tested whether Sir2 becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the heterochromatic HML and HMR loci was not lost during aging. Old cells could initiate a mating response; however, they were less sensitive to mating pheromone than were young cells because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old cells. We propose that aging phenotypes previously attributed to loss of heterochromatin silencing are instead caused by aggregation of the Whi3 cell cycle regulator

Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication. The Origin Recognition Complex (ORC) plays conserved and diverse roles in eukaryotes. Here the authors present the structure of a chromatin interacting domain of yeast Orc1 in complex with the nucleosome core particle, revealing that Orc1 interacts with the histone H4 tail irrespective of K16 acetylation; a modification that regulates accessibility to chromatin.