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Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg 2+, a most-favoured divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg 2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity., (© 2024 Harrell et al.)

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity. Introducing an alkyne at the C18 position showed a similar trend, suggesting tolerance is relatively low even for compact functional groups such as alkynes. Although introduction of a diazirine moiety as a photoreactive group at the C5 position decreased the activity, we found that introducing diazirine at the C13 position was acceptable, and compound 38 exhibited potent NSF activity. These findings will be helpful in the development of molecular probes for NSFs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fungi employ diverse mechanisms for iron uptake to ensure proliferation and survival in iron-limited environments. Siderophores are secondary metabolite small molecules with a high affinity specifically for ferric iron; these molecules play an essential role in iron acquisition in fungi and significantly influence fungal physiology and virulence. Fungal siderophores, which are primarily hydroxamate types, are synthesized via non-ribosomal peptide synthetases (NRPS) or NRPS-independent pathways. Following synthesis, siderophores are excreted, chelate iron, and are transported into the cell by specific cell membrane transporters. In several human pathogenic fungi, siderophores are pivotal for virulence, as inhibition of their synthesis or transport significantly reduces disease in murine models of infection. This review briefly highlights siderophore biosynthesis and transport mechanisms in fungal pathogens as well the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe . Understanding siderophore biosynthesis and transport in pathogenic fungi provides valuable insights into fungal biology and illuminates potential therapeutic targets for combating fungal infections.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are highly conserved endoplasmic reticulum (ER)-resident proteins that establish ER contacts with multiple membrane compartments in many eukaryotes. However, VAP-mediated membrane-tethering mechanisms remain ambiguous. Here, focusing on fission yeast ER-plasma membrane (PM) contact formation, using systematic interactome analyses and quantitative microscopy, we predict a non-VAP-protein direct binding-based ER-PM coupling. We further reveal that VAP-anionic phospholipid interactions may underlie ER-PM association and define the pH-responsive nature of VAP-tethered membrane contacts. Such conserved interactions with anionic phospholipids are generally defective in amyotrophic lateral sclerosis-associated human VAPB mutant. Moreover, we identify a conserved FFAT-like motif locating at the autoinhibitory hotspot of the essential PM proton pump Pma1. This modulatory VAP-Pma1 interaction appears crucial for pH homeostasis. We thus propose an ingenious strategy for maintaining intracellular pH by coupling Pma1 modulation with pH-sensory ER-PM contacts via VAP-mediated interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Cajal body, a nuclear condensate, is crucial for ribonucleoprotein assembly, including small nuclear RNPs (snRNPs). While Coilin has been identified as an integral component of Cajal bodies, its exact function remains unclear. Moreover, no Coilin ortholog has been found in unicellular organisms to date. This study unveils Mug174 (Meiosis-upregulated gene 174) as the Coilin ortholog in the fission yeast Schizosaccharomyces pombe. Mug174 forms phase-separated condensates in vitro and is often associated with the nucleolus and the cleavage body in vivo. The generation of Mug174 foci relies on the trimethylguanosine (TMG) synthase Tgs1. Moreover, Mug174 interacts with Tgs1 and U snRNAs. Deletion of the mug174+ gene in S. pombe causes diverse pleiotropic phenotypes, encompassing defects in vegetative growth, meiosis, pre-mRNA splicing, TMG capping of U snRNAs, and chromosome segregation. In addition, we identified weak homology between Mug174 and human Coilin. Notably, human Coilin expressed in fission yeast colocalizes with Mug174. Critically, Mug174 is indispensable for the maintenance of and transition from cellular quiescence. These findings highlight the Coilin ortholog in fission yeast and suggest that the Cajal body is implicated in cellular quiescence, thereby preventing human diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

3'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing machinery are degenerate and remain poorly characterized compared with the well-defined regulatory elements in mammals. Here we address this issue by developing deep learning models to deconvolute degenerate cis -regulatory elements and quantify their positional importance in mediating yeast poly(A) site formation, cleavage heterogeneity, and strength. In S. cerevisiae , cleavage heterogeneity is promoted by the depletion of U-rich elements around poly(A) sites as well as multiple occurrences of upstream UA-rich elements. Sites with high cleavage heterogeneity show overall lower strength. The site strength and tandem site distances modulate alternative polyadenylation (APA) under the diauxic stress. Finally, we develop a deep learning model to reveal the distinct motif configuration of S. pombe poly(A) sites, which show more precise cleavage than S. cerevisiae Altogether, our deep learning models provide unprecedented insights into poly(A) site formation of yeast species, and our results highlight divergent poly(A) signals across distantly related species., (© 2024 Stroup and Ji; Published by Cold Spring Harbor Laboratory Press.)

Epigenetic mechanisms enable cells to develop novel adaptive phenotypes without altering their genetic blueprint. Recent studies show histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), can be redistributed to establish adaptive phenotypes. We developed a precision-engineered genetic approach to trigger heterochromatin misregulation on-demand in fission yeast. This enabled us to trace genome-scale RNA and H3K9me changes over time in long-term, continuous cultures. Adaptive H3K9me establishes over remarkably slow timescales relative to the initiating stress. We captured dynamic H3K9me redistribution events which depend on an RNA binding complex MTREC, ultimately leading to cells converging on an optimal adaptive solution. Upon stress removal, cells relax to new transcriptional and chromatin states, establishing memory that is tunable and primed for future adaptive epigenetic responses. Collectively, we identify the slow kinetics of epigenetic adaptation that allow cells to discover and heritably encode novel adaptive solutions, with implications for drug resistance and response to infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recombination hotspot-activating DNA sites (e.g., M26 , CCAAT , Oligo-C ) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe . We used a fission yeast-optimized CRISPR-Cas9 system ( SpEDIT ) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6 . PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.

Protein-protein interactions (PPIs) are known to rewire extensively during evolution leading to lineage-specific and species-specific changes in molecular processes. However, the detailed molecular evolutionary mechanisms underlying interactome network rewiring are not well-understood. Here, we combine high-confidence PPI data, high-resolution three-dimensional structures of protein complexes, and homology-based structural annotation transfer to construct structurally-resolved interactome networks for the two yeasts S. cerevisiae and S. pombe. We then classify PPIs according to whether they are preserved or different between the two yeast species and compare site-specific evolutionary rates of interfacial versus non-interfacial residues for these different categories of PPIs. We find that residues in PPI interfaces evolve significantly more slowly than non-interfacial residues when using lineage-specific measures of evolutionary rate, but not when using non-lineage-specific measures. Furthermore, both lineage-specific and non-lineage-specific evolutionary rate measures can distinguish interfacial residues from non-interfacial residues for preserved PPIs between the two yeasts, but only the lineage-specific measure is appropriate for rewired PPIs. Finally, both lineage-specific and non-lineage-specific evolutionary rate measures are appropriate for elucidating structural determinants of protein evolution for residues outside of PPI interfaces. Overall, our results demonstrate that unlike tertiary structures of single proteins, PPIs and PPI interfaces can be highly volatile in their evolution, thus requiring the use of lineage-specific measures when studying their evolution. These results yield insight into the evolutionary design principles of PPIs and the mechanisms by which interactions are preserved or rewired between species, improving our understanding of the molecular evolution of PPIs and PPI interfaces at the residue level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inositol pyrophosphate 1,5-IP 8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1 , pho84 , and tgp1 , via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP 8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP 7 to 1,5-IP 8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 . Aps1 displays a ~twofold preference for hydrolysis of 1-IP 7 versus 5-IP 7 and aps1 ∆ cells have twofold higher levels of 1-IP 7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1 ∆ siw14 ∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP 8 toxicosis, whereby excessive 1,5-IP 8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1 ∆ siw14 ∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1 ∆ siw14 ∆ on YES by deleting tgp1 . However, the severe growth defect of an aps1 ∆ asp1-H397A strain could not be alleviated by deleting tgp1 , suggesting that 1,5-IP 8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity., Importance: Repression of the fission yeast PHO genes tgp1 , pho1 , and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP 8 , a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP 8 is formed by phosphorylation of 5-IP 7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo . Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 in vitro , with a ~twofold preference for 1-IP 7 over 5-IP 7 . aps1 ∆ cells have twofold higher levels of 1-IP 7 than wild-type cells. An aps1 ∆ siw14 ∆ double mutation is lethal because excessive 1,5-IP 8 triggers derepression of tgp1 , leading to toxic uptake of glycerophosphocholine., Competing Interests: The authors declare no conflict of interest.

Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are tightly coordinated to ensure that only fully processed transcripts are released from chromatin for export from the nucleus. Here, we present the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is the key enzyme in an RNP assembly checkpoint at the 3'-end of genes. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localises to cleavage bodies, which are enriched for 3'-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3'-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, and CPAC components are depleted from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites and a delayed transcription termination, suggesting that levels of free CPAC components are insufficient to maintain normal levels of 3'-end processing. Our data support a model in which Dbp2 is the active component of an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release., (© 2024. The Author(s).)

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo . Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.

In the fission yeast Schizosaccharomyces pombe, the response to sulfur depletion has been less studied compared to the response to nitrogen depletion. Our study reveals that the fission yeast gene, SPCC417.09c, plays a significant role in the sulfur depletion response. This gene encodes a protein with a Zn 2 Cys 6 fungal-type DNA-binding domain and a transcription factor domain, and we have named it sdr1 + (sulfur depletion response 1). Interestingly, while sulfur depletion typically induces autophagy akin to nitrogen depletion, we found that autophagy was not induced under sulfur depletion in the absence of sdr1 + . This suggests that sdr1 + is necessary for the induction of autophagy under conditions of sulfur depletion. Although sdr1 + is not essential for the growth of fission yeast, its overexpression, driven by the nmt1 promoter, inhibits growth. This implies that Sdr1 may possess cell growth-inhibitory capabilities. In addition, our analysis of Δsdr1 cells revealed that sdr1 + also plays a role in regulating the expression of genes associated with the phosphate depletion response. In conclusion, our study introduces Sdr1 as a novel transcription factor that contributes to an appropriate cellular nutrient starvation response. It does so by inhibiting inappropriate cell growth and inducing autophagy in response to sulfur depletion., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Centrosomes and spindle pole bodies (SPBs) are important for mitotic spindle formation and serve as cellular signaling platforms. Although centrosomes and SPBs differ in morphology, many mechanistic insights into centrosome function have been gleaned from SPB studies. In the fission yeast Schizosaccharomyces pombe , the α-helical protein Ppc89, identified based on its interaction with the septation initiation network scaffold Sid4, comprises the SPB core. High-resolution imaging has suggested that SPB proteins assemble on the Ppc89 core during SPB duplication, but such interactions are undefined. Here, we define a connection between Ppc89 and the essential pericentrin Pcp1. Specifically, we found that a predicted third helix within Ppc89 binds the Pcp1 pericentrin-AKAP450 centrosomal targeting (PACT) domain complexed with calmodulin. Ppc89 helix 3 contains similarity to p resent i n the N -terminus of C ep57 (PINC) motifs found in the centrosomal proteins fly SAS-6 and human Cep57 and also to the S. cerevisiae SPB protein Spc42. These motifs bind pericentrin-calmodulin complexes and AlphaFold2 models suggest a homologous complex assembles in all four organisms. Mutational analysis of the S. pombe complex supports the importance of Ppc89-Pcp1 binding interface in vivo. Our studies provide insight into the core architecture of the S. pombe SPB and suggest an evolutionarily conserved mechanism of scaffolding pericentrin-calmodulin complexes for mitotic spindle formation., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

TOR protein kinases serve as the catalytic subunit of the TORC1 and TORC2 complexes, which regulate cellular growth, proliferation, and survival. In the fission yeast, Schizosaccharomyces pombe, cells lacking TORC2 or its downstream kinase Gad8 (AKT or SGK1 in human cells) exhibit sensitivity to a wide range of stress conditions, including DNA damage stress. One of the first responses to DNA damage is the phosphorylation of C-terminal serine residues within histone H2AX in human cells (γH2AX), or histone H2A in yeast cells (γH2A). The kinases responsible for γH2A in S. pombe are the two DNA damage checkpoint kinases Rad3 and Tel1 (ATR and ATM, respectively, in human cells). Here we report that TORC2-Gad8 signaling is required for accumulation of γH2A in response to DNA damage and during quiescence. Using the TOR-specific inhibitor, Torin1, we demonstrate that the effect of TORC2 on γH2A in response to DNA damage is immediate, rather than adaptive. The lack of γH2A is restored by deletion mutations of transcription and chromatin modification factors, including loss of components of Paf1C, SAGA, Mediator, and the bromo-domain proteins Bdf1/Bdf2. Thus, we suggest that TORC2-Gad8 may affect the accumulation of γH2A by regulating chromatin structure and function., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Many organisms utilize an actin- and myosin-based cytokinetic ring (CR) to help complete cytokinesis. In Schizosaccharomyces pombe , the Septation Initiation Network (SIN) promotes proper CR function and stability. The SIN is a conserved and essential signaling network consisting of a GTPase and a cascade of kinases assembled at the spindle pole body (SPB). The PP2A SIN inhibitory phosphatase (SIP) complex related to the STRIPAK phosphatase complex is one inhibitor of SIN signaling. The SIP consists of Csc1, Csc2, Csc3, Csc4, Paa1, and the phosphatase subunit Ppa3. Here, we determine that the SIP is anchored at the SPB via the Csc1 FHA domain and that constitutive SPB localization of the SIP is lethal due to persistent SIN inhibition. Disrupting SIP docking at the SPB with a point mutation within the FHA domain or eliminating phosphatase activity by introducing a point mutation within Ppa3 resulted in intact SIP complexes without SIN inhibitory function. Lastly, we defined the unique features of Ppa3 that allow it, but not two other PP2A catalytic subunits, to incorporate into the SIP. Overall, we provide insight into how the SIP complex assembles, localizes, and functions to counteract the SIN with spatiotemporal precision during cytokinesis.

HP1 proteins are essential for establishing and maintaining transcriptionally silent heterochromatin. They dimerize, forming a binding interface to recruit diverse chromatin-associated factors. Although HP1 proteins are known to rapidly evolve, the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impacts heterochromatin formation, we performed a targeted mutagenesis screen of the S. pombe HP1 homolog, Swi6. Substitutions within an auxiliary surface adjacent to the HP1 dimerization interface produce Swi6 variants with divergent maintenance properties. Remarkably, substitutions at a single amino acid position lead to the persistent gain or loss of epigenetic inheritance. These substitutions increase Swi6 chromatin occupancy in vivo and altered Swi6-protein interactions that reprogram H3K9me maintenance. We show how relatively minor changes in Swi6 amino acid composition in an auxiliary surface can lead to profound changes in epigenetic inheritance providing a redundant mechanism to evolve HP1-effector specificity., (© 2024. The Author(s).)

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3' end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1 , pho84 , and tgp1 ) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP 8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP 8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP 8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP 8 toxicity is alleviated by deletion of snf22 + , the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22- ( D996A-E997A ) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22 + hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO -repressive lncRNAs. Transcription profiling of snf22 ∆ and snf22- ( D996A-E997A ) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1 , pho1 , and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP 8 as an agonist of precocious lncRNA 3'-processing/termination. IP 8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP 8 levels on termination. In this study, a forward genetic screen revealed that IP 8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO -activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters., Competing Interests: The authors declare no conflict of interest.

Cells switch genes ON or OFF by altering the state of chromatin via histone modifications at specific regulatory locations along the chromatin polymer. These gene regulation processes are carried out by a network of reactions in which the histone marks spread to neighboring regions with the help of enzymes. In the literature, this spreading has been studied as a purely kinetic, non-diffusive process considering the interactions between neighboring nucleosomes. In this work, we go beyond this framework and study the spreading of modifications using a reaction-diffusion (RD) model accounting for the diffusion of the constituents. We quantitatively segregate the modification profiles generated from kinetic and RD models. The diffusion and degradation of enzymes set a natural length scale for limiting the domain size of modification spreading, and the resulting enzyme limitation is inherent in our model. We also demonstrate the emergence of confined modification domains without the explicit requirement of a nucleation site. We explore polymer compaction effects on spreading and show that single-cell domains may differ from averaged profiles. We find that the modification profiles from our model are comparable with existing H3K9me3 data of S. pombe., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Manivannan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Telomeric repeat-binding factor 1 (Tbf1) has a similar architecture as the TRF family of telomeric proteins and plays important roles in both telomere homeostasis and ribosome regulation. However, the molecular basis of why Tbf1 has such different functions compared to other TRFs remains unclear. Here, we present the crystal structures of the TRF homology (TRFH) and Myb-L domains from Schizosaccharomyces pombe Tbf1 (spTbf1). TRFH-mediated homodimerization is essential for spTbf1 stability. Importantly, spTbf1 TRFH lacks the conserved docking motif for interactions with telomeric proteins, explaining why spTbf1 does not participate in the assembly of the shelterin complex. Finally, structural and biochemical analyses demonstrate that TRFH and Myb-L domains as well as the loop region of spTbf1 coordinate to recognize S. pombe telomeric double-stranded DNA. Overall, our findings provide structural and functional insights into how fungi Tbf1 acts as an atypical telomeric repeat-binding factor, which helps to understand the evolution of TRFH-containing telomeric proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (nonsister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genome-wide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1Δ and rif1Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1+ deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1+ separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis., Competing Interests: Conflicts of interest The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. Nevan Krogan has a financially compensated consulting agreement with Maze Therapeutics. Nevan is the President and is on the Board of Directors of Rezo Therapeutics, and he is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cockrell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

During meiosis, defects in cohesin localization within the centromere region can result in various diseases. Accurate cohesin localization depends on the Mis4-Ssl3 loading complex. Although it is known that cohesin completes the loading process with the help of the loading complex, the mechanisms underlying its localization in the centromere region remain unclear. Previous studies suggest cohesin localization in the centromere is mediated by phosphorylation of centromeric proteins. In this study, we focused on the Fta2 protein, a component of the Sim4 centromere protein complex. Using bioinformatics methods, potential phosphorylation sites were identified, and fta2-9A and fta2-9D mutants were constructed in Schizosaccharomyces pombe . The phenotypes of these mutants were characterized through testing thiabendazole (TBZ) sensitivity and fluorescent microscopy localization. Results indicated that Fta2 phosphorylation did not impact mitosis but affected chromosome segregation during meiosis. This study suggests that Fta2 phosphorylation is vital for meiosis and may be related to the specific localization of cohesin during this process.

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1 + disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Ribosomes mediate protein synthesis, which is one of the most energy-demanding activities within the cell, and mitochondria are one of the main sources generating energy. How mitochondrial morphology and functions are adjusted to cope with ribosomal defects, which can impair protein synthesis and affect cell viability, is poorly understood. Here, we used the fission yeast Schizosaccharomyces Pombe as a model organism to investigate the interplay between ribosome and mitochondria. We found that a ribosomal insult, caused by the absence of Rpl2702, activates a signaling pathway involving Sty1/MAPK and mTOR to modulate mitochondrial morphology and functions. Specifically, we demonstrated that Sty1/MAPK induces mitochondrial fragmentation in a mTOR-independent manner while both Sty1/MAPK and mTOR increases the levels of mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS). Moreover, we demonstrated that Sty1/MAPK acts upstream of Tor1/TORC2 and Tor1/TORC2 and is required to activate Tor2/TORC1. The enhancements of mitochondrial membrane potential and mROS function to promote proliferation of cells bearing ribosomal defects. Hence, our study reveals a previously uncharacterized Sty1/MAPK-mTOR signaling axis that regulates mitochondrial morphology and functions in response to ribosomal insults and provides new insights into the molecular and physiological adaptations of cells to impaired protein synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nitric oxide (˙NO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ˙NO-mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S-nitrosoglutathione reductase Fmd2. To investigate heme-dependent activation of Yhb1 in response to ˙NO, we use hem1Δ-derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1 + mRNA levels are repressed in the presence of iron through a mechanism involving the GATA-type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1 + is derepressed and further induced in the presence of the ˙NO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin-dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin-dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Schizosaccharomyces pombe (fission yeast) is an attractive model for mitochondrial research. The organism resembles human cells in terms of mitochondrial inheritance, mitochondrial transport, sugar metabolism, mitogenome structure and dependence of viability on the mitogenome (the petite-negative phenotype). Transcriptions of these genomes produce only a few polycistronic transcripts, which then undergo processing as per the tRNA punctuation model. In general, the machinery for mitochondrial gene expression is structurally and functionally conserved between fission yeast and humans. Furthermore, molecular research on S. pombe is supported by a considerable number of experimental techniques and database resources. Owing to these advantages, fission yeast has significantly contributed to biomedical and fundamental research. Here, we review the current state of knowledge regarding S. pombe mitochondrial gene expression, and emphasise the pertinence of fission yeast as both a model and tool, especially for studies on mitochondrial translation., (© 2023 The Authors. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Calcineurin (CN) is a conserved Ca 2+ /calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca 2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl - -homeostasis, and Ppb1 deletion induces MgCl 2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1 + . Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl 2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca 2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1 + causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl 2 in wild-type cells, the arsenite tolerance, but not MgCl 2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl - homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested that changes in the biophysical properties of the cytoplasm play a key role in cellular homeostasis and adaptation. However, it still remains unclear how cells control their cytoplasmic properties in response to environmental cues. Here, we used fission yeast spores as a model system of dormant cells to elucidate the mechanisms underlying regulation of the cytoplasmic properties. By tracking fluorescent tracer particles, we found that particle mobility decreased in spores compared to vegetative cells and rapidly increased at the onset of dormancy breaking upon glucose addition. This cytoplasmic fluidization depended on glucose-sensing via the cyclic adenosine monophosphate-protein kinase A pathway. PKA activation led to trehalose degradation through trehalase Ntp1, thereby increasing particle mobility as the amount of trehalose decreased. In contrast, the rapid cytoplasmic fluidization did not require de novo protein synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, the measurement of diffusion coefficients with tracer particles of different sizes suggests that the spore cytoplasm impedes the movement of larger protein complexes (40 to 150 nm) such as ribosomes, while allowing free diffusion of smaller molecules (~3 nm) such as second messengers and signaling proteins. Our experiments have thus uncovered a series of signaling events that enable cells to quickly fluidize the cytoplasm at the onset of dormancy breaking., Competing Interests: Competing interests statement:The authors declare no competing interest.

Movement dynamics in the nucleus involve various biological processes, including DNA repair, which is crucial for cancer prevention. Changes in the movement of the components of the nucleus indicate the changes in movement dynamics in the nucleus. In Schizosaccharomyces pombe, the inner nuclear membrane protein Bqt4 plays an essential role in attaching telomeres to the nuclear envelope. We observed that the deletion of bqt4 + caused a significant decrease in the mean square displacement (MSD) calculated from the distance between the nucleolar center and spindle pole body (SPB), hereafter referred to as MSD (SPB-Nucleolus) . The MSD (SPB-Nucleolus) decrease in bqt4Δ was microtubule-dependent. The Rap1-binding ability loss mutant, bqt4 F46A , and nonspecific DNA-binding ability mutants, bqt4 3E-A , did not exhibit an MSD (SPB-Nucleolus) decrease compared to the WT. Moreover, the bqt4 3E-A rap1Δ double mutant and 1-262 amino acids truncated mutant bqt4ΔN (263-432), which does not have either Rap1-binding or nonspecific DNA-binding abilities, did not exhibit the MSD(SPB-Nucleolus) decrease to the same extent as bqt4Δ. These results suggest that the unknown function of Bqt4 in the C-terminal domain is essential for the maintenance of the pattern of relative movement between SPB and the nucleolus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Byr2 kinase of fission yeast Schizosaccharomyces pombe is recruited to the membrane with the assistance of Ras1. Byr2 is also negatively regulated by 14-3-3 proteins encoded by rad24 and rad25. We conducted domain and mutational analysis of Byr2 to determine which region is critical for its binding to 14-3-3 proteins. Rad24 and Rad25 bound to both the Ras interaction domain in the N-terminus and to the C-terminal catalytic domain of Byr2. When amino acid residues S87 and T94 of the Ras-interacting domain of Byr2 were mutated to alanine, Rad24 could no longer bind to Byr2. S402, S566, S650, and S654 mutations in the C-terminal domain of Byr2 also abolished its interaction with Rad24 and Rad25. More than three mutations in the C-terminal domain were required to abolish completely its interaction with 14-3-3 protein, suggesting that multiple residues are involved in this interaction. Expression of the N-terminal domain of Byr2 in wild-type cells lowered the mating ratio, because it likely blocked the interaction of Byr2 with Ste4 and Ras1, whereas expression of the catalytic domain of Byr2 increased the mating ratio as a result of freeing from intramolecular regulation by the N-terminal domain of Byr2. The S87A and T94A mutations of Byr2 increased the mating ratio and attenuated inhibition of Byr2 by Rad24; therefore, these two amino acids are critical for its regulation by Rad24. S566 of Byr2 is critical for activity of Byr2 but not for its interaction with 14-3-3 proteins. In this study, we show that 14-3-3 proteins interact with two separate domains in Byr2 as negative regulators., (© 2024. The Author(s).)

Alcohol fermentation comprises two phases: phase 1, alcohol fermentation occurs while yeast cells proliferate; phase 2, growth stops and alcohol fermentation continues. We categorized genes related to proliferation in low ethanol (phase 1) and viability in high ethanol (phase 2) as Alcohol Growth Ability (AGA) and Alcohol Viability (ALV), respectively. Although genes required for phase 1 are examined in budding yeast, those for phase 2 are unknown. We set conditions for ALV screening, searched for protein kinases (PKs) related to ALV in budding yeast, and expanded two screenings to fission yeast. Bub1 kinase was important for proliferation in low ethanol but not for viability in high ethanol, suggesting that the important PKs differ between the two phases. It was indeed the case. Further, 3 common PKs were identified as AGA in both yeasts, suggesting that the important cellular mechanism in phase 1 is conserved in both yeasts, at least partially., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers. For complete details on the use and execution of this protocol, please refer to Tang et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Baranowska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of interests M.M. and J.S.K. have a pending US provisional patent application related to the data from this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A new study reports the identification of a fission yeast dynamin superfamily protein, Mmc1, that self-assembles on the matrix side of the inner mitochondrial membrane and interacts with subunits of the mitochondrial contact site and cristae organizing system to maintain cristae architecture., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial contact site and cristae organizing system (MICOS) complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae-shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS to promote normal mitochondrial morphology and respiratory function. Mmc1 is a distant relative of the dynamin superfamily of proteins (DSPs), GTPases, which are well established to shape and remodel membranes. Similar to DSPs, Mmc1 self-associates and forms high-molecular-weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting that it does not dynamically remodel membranes. These data are consistent with the model that Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pathogenic bacteria employ virulence factors (VF) to establish infection and cause disease in their host. Yeasts, Saccharomyces cerevisiae and Saccharomyces pombe, are useful model organisms to study the functions of bacterial VFs and their interaction with targeted cellular processes because yeast processes and organelle structures are highly conserved and similar to higher eukaryotes. In this review, we describe the principles and applications of the yeast model for the identification and functional characterisation of bacterial VFs to investigate bacterial pathogenesis. The growth inhibition phenotype caused by the heterologous expression of bacterial VFs in yeast is commonly used to identify candidate VFs. Then, subcellular localisation patterns of bacterial VFs can provide further clues about their target molecules and functions during infection. Yeast knockout and overexpression libraries are also used to investigate VF interactions with conserved eukaryotic cell structures (e.g., cytoskeleton and plasma membrane), and cellular processes (e.g., vesicle trafficking, signalling pathways, and programmed cell death). In addition, the yeast growth inhibition phenotype is also useful for screening new drug leads that target and inhibit bacterial VFs. This review provides an updated overview of new tools, principles and applications to study bacterial VFs in yeast., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Borao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants., (© 2024. The Author(s).)

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm., (© 2024. The Author(s).)

In fission yeast, Schizosaccharomyces pombe, constitutive heterochromatin defined by methylation of histone H3 lysine 9 (H3K9me) and its binding protein Swi6/HP1 localizes at the telomere, centromere, and mating-type loci. These loci contain DNA sequences called dg and dh, and the RNA interference (RNAi)-dependent system establishes and maintains heterochromatin at dg/dh. Bi-directional transcription at dg/dh induced by RNA polymerase II is critical in RNAi-dependent heterochromatin formation because the transcribed RNAs provide substrates for siRNA synthesis and a platform for assembling RNAi factors. However, a regulator of dg/dh transcription during the establishment of heterochromatin is not known. Here, we found that a zinc-finger protein Moc3 localizes dh and activates dh-forward transcription in its zinc-finger-dependent manner when heterochromatin structure or heterochromatin-dependent silencing is compromised. However, Moc3 does not localize at normal heterochromatin and does not activate the dh-forward transcription. Notably, the loss of Moc3 caused a retarded heterochromatin establishment, showing that Moc3-dependent dh-forward transcription is critical for RNAi-dependent heterochromatin establishment. Therefore, Moc3 is a transcriptional activator that induces RNAi to establish heterochromatin., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in 'competition' reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)