RESUMEN
Transposable elements (TEs) are ubiquitous in the eukaryote genomes, but their evolutionary and functional significance remains largely obscure and contentious. Here, we explore the evolution and functional impact of TEs in two model unicellular eukaryotes, the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which diverged around 330 to 420 million years ago. We analyze the distribution of LTR retrotransposons (LTR-RTs, the only TE order identified in both species) and their solo-LTR derivatives in 35 strains of S. pombe and 128 strains of S. cerevisiae. We find that natural LTR-RT and solo-LTR insertions exhibit high presence-absence polymorphism among individuals in both species. Population genetics analyses show that solo-LTR insertions experienced functional constraints similar to synonymous sites of host genes in both species, indicating a majority of solo-LTR insertions might have evolved in a neutral manner. When knocking out nine representative solo-LTR insertions separately in the S. pombe strain 972h- and 12 representative solo-LTR insertions separately in the S. cerevisiae strain S288C, we find that one solo-LTR insertion in S. pombe has a significant effect on the fitness and transcriptome of its host. Together, our findings indicate that a fraction of natural TE insertions likely shape their host transcriptomes and thereby contribute to their host fitness, with implications for understanding the functional significance of TEs in eukaryotes.
Asunto(s)
Aptitud Genética , Retroelementos , Saccharomyces cerevisiae , Schizosaccharomyces , Schizosaccharomyces/genética , Saccharomyces cerevisiae/genética , Elementos Transponibles de ADN , Evolución Molecular , Secuencias Repetidas Terminales , Mutagénesis Insercional , Genoma FúngicoRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Meiosis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Meiosis/genética , Sistemas CRISPR-Cas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Recombinación Genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Edición Génica/métodosRESUMEN
With the widespread adoption of next-generation sequencing technologies, the speed and convenience of genome sequencing have significantly improved, and many biological genomes have been sequenced. However, during the assembly of small genomes, we still face a series of challenges, including repetitive fragments, inverted repeats, low sequencing coverage, and the limitations of sequencing technologies. These challenges lead to unknown gaps in small genomes, hindering complete genome assembly. Although there are many existing assembly software options, they do not fully utilize the potential of artificial intelligence technologies, resulting in limited improvement in gap filling. Here, we propose a novel method, DLGapCloser, based on deep learning, aimed at assisting traditional tools in further filling gaps in small genomes. Firstly, we created four datasets based on the original genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla. To further extract effective information from the gene sequences, we also added homologous genomes to enrich the datasets. Secondly, we proposed the DGCNet model, which effectively extracts features and learns context from sequences flanking gaps. Addressing issues with early pruning and high memory usage in the Beam Search algorithm, we developed a new prediction algorithm, Wave-Beam Search. This algorithm alternates between expansion and contraction phases, enhancing efficiency and accuracy. Experimental results showed that the Wave-Beam Search algorithm improved the gap-filling performance of assembly tools by 7.35%, 28.57%, 42.85%, and 8.33% on the original results. Finally, we established new gap-filling standards and created and implemented a novel evaluation method. Validation on the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla showed that DLGapCloser increased the number of filled gaps by 8.05%, 15.3%, 1.4%, and 7% compared to traditional assembly tools.
Asunto(s)
Redes Neurales de la Computación , Algoritmos , Aprendizaje Profundo , Genoma Fúngico , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neurospora crassa/genética , Programas Informáticos , Genómica/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
During meiosis, transient associations between the nuclear envelope and telomeres transmit nuclear movements to chromosomes, enabling their pairing and recombination. Recent advances in the field of quantitative cell biology allow a large volume of information about the kinetics of these chromosome movements to be extracted and analyzed with the aim of identifying biologically relevant movement patterns. To this end, we have developed ChroMo, a freely available application for the unsupervised study of chromosome movements in fission yeast meiosis. ChroMo contains a set of time series algorithms to identify chromosome movement motifs that are not easily observable by direct human visualization and to establish causal relationships between phenotypes. In this chapter, we present a detailed protocol for the processing of raw live imaging data from fission yeast and its subsequent analysis in ChroMo.
Asunto(s)
Cromosomas Fúngicos , Meiosis , Schizosaccharomyces , Schizosaccharomyces/genética , Cromosomas Fúngicos/genética , Algoritmos , Telómero/genética , Telómero/metabolismo , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions.
Asunto(s)
Daño del ADN , Diploidia , Regulación Fúngica de la Expresión Génica , Haploidia , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/efectos de los fármacos , Metilmetanosulfonato/farmacología , Transcriptoma , Transcripción Genética , Perfilación de la Expresión Génica , Mutágenos/toxicidad , Mutágenos/farmacologíaRESUMEN
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.
Asunto(s)
Epigénesis Genética , Heterocromatina , Histonas , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Heterocromatina/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Histonas/genética , Adaptación Fisiológica/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Regulación Fúngica de la Expresión Génica , MetilaciónRESUMEN
Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1's involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.
Asunto(s)
Replicación del ADN , Epigénesis Genética , Histonas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa I/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Histonas/genética , Unión Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.
Asunto(s)
Replicación del ADN , Epigénesis Genética , Histonas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Histonas/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Mutación , Memoria EpigenéticaRESUMEN
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 Zn2Cys6 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.
Asunto(s)
Autofagia , Regulación Fúngica de la Expresión Génica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Azufre , Factores de Transcripción , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Azufre/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regiones Promotoras GenéticasRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona , Meiosis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Unión Proteica , Cinetocoros/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Técnicas del Sistema de Dos Híbridos , Segregación Cromosómica , Cohesinas , FosfoproteínasRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box , Ribonucleoproteínas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Poliadenilación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Cromatina/metabolismo , ARN de Hongos/metabolismo , ARN de Hongos/genética , Núcleo Celular/metabolismoRESUMEN
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.
Asunto(s)
Daño del ADN , Histonas , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Histonas/metabolismo , Histonas/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/genética , Transducción de Señal , Fosforilación , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Humanos , Proteínas Serina-Treonina QuinasasRESUMEN
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.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Polaridad Celular , Endocitosis , Retroalimentación Fisiológica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteína de Unión al GTP cdc42 , Quinasas p21 Activadas , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/genética , Actinas/metabolismoRESUMEN
Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.
Asunto(s)
Adenosina Desaminasa , Edición de ARN , Proteínas de Unión al ARN , Schizosaccharomyces , Schizosaccharomyces/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Edición de ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Humanos , Especificidad por Sustrato , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Adenosina/metabolismo , Adenosina/genética , Inosina/genética , Inosina/metabolismoRESUMEN
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 present in the N-terminus of Cep57 (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.
Asunto(s)
Centrosoma , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Huso Acromático , Cuerpos Polares del Huso , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Cuerpos Polares del Huso/metabolismo , Centrosoma/metabolismo , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos/metabolismo , Calmodulina/metabolismo , Unión ProteicaRESUMEN
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.
Asunto(s)
ADN Ribosómico , Ribosomas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , ADN Ribosómico/genética , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Regulación Fúngica de la Expresión Génica , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Transducción de Señal/genética , Ciclo Celular/genética , Eliminación de GenRESUMEN
Traditionally, hybrid promoters are constructed, in Saccharomyces cerevisiae, by joining the core region and the upstream activating sequences from different native promoters. Here, we describe a new design that makes use of the core promoters from foreign organisms: viruses, humans, and the yeast Schizosaccharomyces pombe. With this approach, we realized a library of 59 new constitutive promoters that span over nine folds in gene expression.
Asunto(s)
Regiones Promotoras Genéticas , Saccharomyces cerevisiae , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Biblioteca de Genes , Schizosaccharomyces/genética , Humanos , Ingeniería Genética/métodosRESUMEN
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.
Asunto(s)
Meiosis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/efectos de los fármacos , Cohesinas , Meiosis/efectos de los fármacos , Fosforilación , Schizosaccharomyces/citología , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona , Epigénesis Genética , Heterocromatina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Homólogo de la Proteína Chromobox 5 , Histonas/metabolismo , Histonas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Unión Proteica , Cromatina/metabolismo , Memoria EpigenéticaRESUMEN
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.