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1.
BMC Bioinformatics ; 21(1): 478, 2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33099301

RESUMO

BACKGROUND: Introns have been shown to be spliced in a defined order, and this order influences both alternative splicing regulation and splicing fidelity, but previous studies have only considered neighbouring introns. The detailed intron splicing order remains unknown. RESULTS: In this work, a method was developed that can calculate the intron splicing orders of all introns in each transcript. A simulation study showed that this method can accurately calculate intron splicing orders. I further applied this method to real S. pombe, fruit fly, Arabidopsis thaliana, and human sequencing datasets and found that intron splicing orders change from gene to gene and that humans contain more not in-order spliced transcripts than S. pombe, fruit fly and Arabidopsis thaliana. In addition, I reconfirmed that the first introns in humans are spliced slower than those in S. pombe, fruit fly, and Arabidopsis thaliana genome-widely. Both the calculated most likely orders and the method developed here are available on the web. CONCLUSIONS: A novel computational method was developed to calculate the intron splicing orders and applied the method to real sequencing datasets. I obtained intron splicing orders for hundreds or thousands of genes in four organisms. I found humans contain more number of not in-order spliced transcripts.


Assuntos
Arabidopsis/genética , Biologia Computacional/métodos , Drosophila melanogaster/genética , Íntrons/genética , Processamento de RNA/genética , Schizosaccharomyces/genética , Processamento Alternativo , Animais , Sequência de Bases , Humanos
2.
Nucleic Acids Res ; 48(19): 10998-11015, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045725

RESUMO

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.


Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , DNA Fúngico/química , Quadruplex G , Schizosaccharomyces/genética , DNA Helicases/fisiologia , Compostos de Anéis Fundidos/farmacologia , Fase S , Proteínas de Schizosaccharomyces pombe/fisiologia
3.
Nature ; 585(7823): 119-123, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848252

RESUMO

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.


Assuntos
Mitose , Membrana Nuclear/metabolismo , Schizosaccharomyces/citologia , Divisão Celular , Modelos Biológicos , Poro Nuclear/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura
4.
Mol Cell ; 79(6): 963-977.e3, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32735772

RESUMO

Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Proteínas R-SNARE/genética , Autofagossomos/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Proteólise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Resposta a Proteínas não Dobradas/genética
5.
Proc Natl Acad Sci U S A ; 117(35): 21504-21511, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817556

RESUMO

In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.


Assuntos
Proteínas de Ligação a DNA/genética , Heterocromatina/metabolismo , Sequências Repetidas Invertidas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica/fisiologia , Heterocromatina/genética , Histonas/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo
6.
Nucleic Acids Res ; 48(15): 8243-8254, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32720681

RESUMO

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes - pho1, pho84, and tgp1 - that are repressed under phosphate-replete conditions by 5' flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor 'precocious' lncRNA 3'-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Longo não Codificante/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Transcrição Genética , Homeostase , Nutrientes/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , RNA Mensageiro , Regulon , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
7.
Nucleic Acids Res ; 48(16): 8977-8992, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32710633

RESUMO

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.


Assuntos
Motivos de Nucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Acetiltransferases/metabolismo , Regulação Fúngica da Expressão Gênica , Histidina/metabolismo , Fases de Leitura Aberta , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , eIF-2 Quinase/metabolismo
8.
PLoS Genet ; 16(7): e1008933, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692737

RESUMO

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Fase S/genética , Schizosaccharomyces/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
9.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
10.
Genes Dev ; 34(13-14): 883-897, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32499400

RESUMO

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , RNA Polimerase II/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Ativação Enzimática/efeitos dos fármacos , Genes Fúngicos/genética , Mutação , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fosfatos/farmacologia , Poliadenilação , Regiões Promotoras Genéticas/genética , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
11.
Mol Cell ; 79(3): 488-503.e11, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32585128

RESUMO

Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.


Assuntos
RNA Polimerase III/genética , RNA Polimerase II/genética , RNA Polimerase I/genética , RNA Fúngico/química , Saccharomyces cerevisiae/genética , Elongação da Transcrição Genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , Dobramento de RNA , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Sítios de Splice de RNA , Processamento de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Termodinâmica
12.
RNA ; 26(10): 1380-1388, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32513655

RESUMO

Pat1, known as Pdc2 in fission yeast, promotes the activation and assembly of multiple proteins during mRNA decay. After deadenylation, the Pat1/Lsm1-7 complex binds to transcripts containing oligo(A) tails, which can be modified by the addition of several terminal uridine residues. Pat1 enhances Lsm1-7 binding to the 3' end, but it is unknown how this interaction is influenced by nucleotide composition. Here we examine Pat1/Lsm1-7 binding to a series of oligoribonucleotides containing different A/U contents using recombinant purified proteins from fission yeast. We observe a positive correlation between fractional uridine content and Lsm1-7 binding affinity. Addition of Pat1 broadens RNA specificity of Lsm1-7 by enhancing binding to A-rich RNAs and increases cooperativity on all oligonucleotides tested. Consistent with increased cooperativity, Pat1 promotes multimerization of the Lsm1-7 complex, which is potentiated by RNA binding. Furthermore, the inherent ability of Pat1 to multimerize drives liquid-liquid phase separation with multivalent decapping enzyme complexes of Dcp1/Dcp2. Our results uncover how Pat1 regulates RNA binding and higher order assembly by mRNA decay factors.


Assuntos
Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Fatores de Transcrição/genética , Citoplasma/genética , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética
13.
RNA ; 26(10): 1334-1344, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32546512

RESUMO

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.


Assuntos
Fosfatase Ácida/genética , Proteínas de Transporte/genética , Regulação Fúngica da Expressão Gênica/genética , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Terminação da Transcrição Genética/fisiologia , Fosfatos de Inositol/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Mensageiro/genética
14.
Nucleic Acids Res ; 48(13): 7154-7168, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496538

RESUMO

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Histonas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Elongação da Transcrição Genética , Fosforilação , Fatores de Elongação da Transcrição/metabolismo , Ubiquitinação
15.
Nucleic Acids Res ; 48(10): 5788-5798, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32374858

RESUMO

The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Desoxirribonucleases/metabolismo , Francisella/enzimologia , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Polimerase II/metabolismo , Ribonucleases/metabolismo , Schizosaccharomyces/genética
16.
Nat Commun ; 11(1): 2412, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415063

RESUMO

Long non-coding RNAs (lncRNAs) are components of epigenetic control mechanisms that ensure appropriate and timely gene expression. The functions of lncRNAs are often mediated through associated gene regulatory activities, but how lncRNAs are distinguished from other RNAs and recruit effector complexes is unclear. Here, we utilize the fission yeast Schizosaccharomyces pombe to investigate how lncRNAs engage silencing activities to regulate gene expression in cis. We find that invasion of lncRNA transcription into the downstream gene body incorporates a cryptic intron required for repression of that gene. Our analyses show that lncRNAs containing cryptic introns are targeted by the conserved Pir2ARS2 protein in association with splicing factors, which recruit RNA processing and chromatin-modifying activities involved in gene silencing. Pir2 and splicing machinery are broadly required for gene repression. Our finding that human ARS2 also interacts with splicing factors suggests a conserved mechanism mediates gene repression through cryptic introns within lncRNAs.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Íntrons , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Processamento Alternativo , Cromatina/metabolismo , Cruzamentos Genéticos , Inativação Gênica , Genoma Fúngico , Proteínas de Choque Térmico/genética , Proteínas Nucleares/genética , Interferência de RNA , Sítios de Splice de RNA , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , RNA-Seq , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 117(22): 12062-12070, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414915

RESUMO

Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/fisiologia , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , DNA/metabolismo , Meiose/fisiologia , Recombinases Rec A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo
18.
Nat Commun ; 11(1): 2447, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415081

RESUMO

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.


Assuntos
Citosina/metabolismo , DNA Fúngico/genética , Desoxiadenosinas/metabolismo , Genoma Fúngico , Guanosina/metabolismo , Ribonucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Núcleo Celular/genética , DNA Mitocondrial/genética , Genoma Mitocondrial , Sequências Repetitivas de Ácido Nucleico/genética , Ribonuclease H/metabolismo , Schizosaccharomyces/genética
19.
PLoS Genet ; 16(4): e1008330, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324744

RESUMO

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.


Assuntos
Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alelos , Anticódon , Citosol/enzimologia , Teste de Complementação Genética , Humanos , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Alinhamento de Sequência , Especificidade por Substrato
20.
Nat Commun ; 11(1): 1973, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332728

RESUMO

The genetics of quiescence is an emerging field compared to that of growth, yet both states generate spontaneous mutations and genetic diversity fueling evolution. Reconciling mutation rates in dividing conditions and mutation accumulation as a function of time in non-dividing situations remains a challenge. Nitrogen-starved fission yeast cells reversibly arrest proliferation, are metabolically active and highly resistant to a variety of stresses. Here, we show that mutations in stress- and mitogen-activated protein kinase (S/MAPK) signaling pathways are enriched in aging cultures. Targeted resequencing and competition experiments indicate that these mutants arise in the first month of quiescence and expand clonally during the second month at the expense of the parental population. Reconstitution experiments show that S/MAPK modules mediate the sacrifice of many cells for the benefit of some mutants. These findings suggest that non-dividing conditions promote genetic diversity to generate a social cellular environment prone to kin selection.


Assuntos
Sistema de Sinalização das MAP Quinases , Mitose , Mutação , Nitrogênio/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Técnicas de Cocultura , DNA/metabolismo , Citometria de Fluxo , Variação Genética , Genótipo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Análise de Sequência de DNA , Transdução de Sinais , Processos Estocásticos
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