RESUMO
Telomeres form the ends of linear chromosomes and protect these ends from being recognized as DNA double-strand breaks. Telomeric sequences are maintained in most cells by telomerase, a reverse transcriptase that adds TG-rich repeats to chromosome ends. In budding yeast, telomeres are organized in clusters at the nuclear periphery by interactions that depend on components of silent chromatin and the telomerase-binding factor yeast Ku (yKu). In this study, we examined whether the subnuclear localization of telomeres affects end maintenance. A telomere anchoring pathway involving the catalytic yeast telomerase subunits Est2, Est1, and Tlc1 is shown to be necessary for the perinuclear anchoring activity of Yku80 during S phase. Additionally, we identify the conserved Sad1-UNC-84 (SUN) domain protein Mps3 as the principal membrane anchor for this pathway. Impaired interference with Mps3 anchoring through overexpression of the Mps3 N terminus in a tel1 deletion background led to a senescence phenotype and to deleterious levels of subtelomeric Y' recombination. This suggests that telomere binding to the nuclear envelope helps protect telomeric repeats from recombination. Our results provide an example of a specialized structure that requires proper spatiotemporal localization to fulfill its biological role, and identifies a novel pathway of telomere protection.
Assuntos
Proteínas de Membrana/metabolismo , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares , Ligação Proteica , Recombinação Genética/genética , Fase S/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3' untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas/genética , Galactose/metabolismo , Galactose/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hexoquinase/genética , Isoenzimas/genética , Poro Nuclear/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Telômeros/análise , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Budding yeast telomeres are reversibly bound at the nuclear envelope through two partially redundant pathways that involve the Sir2/3/4 silencing complex and the Yku70/80 heterodimer. To better understand how this is regulated, we studied the role of SUMOylation in telomere anchoring. We find that the PIAS-like SUMO E3 ligase Siz2 sumoylates both Yku70/80 and Sir4 in vivo and promotes telomere anchoring to the nuclear envelope. Remarkably, loss of Siz2 also provokes telomere extension in a telomerase-dependent manner that is epistatic with loss of the helicase Pif1. Consistent with our previously documented role for telomerase in anchorage, normal telomere anchoring in siz2 Δ is restored by PIF1 deletion. By live-cell imaging of a critically short telomere, we show that telomeres shift away from the nuclear envelope when elongating. We propose that SUMO-dependent association with the nuclear periphery restrains bound telomerase, whereas active elongation correlates with telomere release.
Assuntos
Membrana Nuclear/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Telômero/enzimologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Microscopia de Fluorescência , Mutação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sumoilação , Fatores de TempoRESUMO
The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure.
Assuntos
Biologia/métodos , Núcleo Celular/metabolismo , Saccharomycetales/fisiologia , Nucléolo Celular/ultraestrutura , Cromatina/metabolismo , Cromossomos/ultraestrutura , Reparo do DNA , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mitose , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Recombinação Genética , Telômero/ultraestruturaRESUMO
RNaseIII ribonucleases act at the heart of RNA silencing pathways by processing precursor RNAs into mature microRNAs and siRNAs. In the fission yeast Schizosaccharomyces pombe, siRNAs are generated by the RNaseIII enzyme Dcr1 and are required for heterochromatin formation at centromeres. In this study, we have analyzed the subcellular localization of Dcr1 and found that it accumulates in the nucleus and is enriched at the nuclear periphery. Nuclear accumulation of Dcr1 depends on a short motif that impedes nuclear export promoted by the double-stranded RNA binding domain of Dcr1. Absence of this motif renders Dcr1 mainly cytoplasmic and is accompanied by remarkable changes in gene expression and failure to assemble heterochromatin. Our findings suggest that Dicer proteins are shuttling proteins and that the steady-state subcellular levels can be shifted toward either compartment.
Assuntos
Núcleo Celular/genética , Heterocromatina/genética , Interferência de RNA/fisiologia , Ribonuclease III/metabolismo , Schizosaccharomyces/genética , Transporte Ativo do Núcleo Celular/genética , Motivos de Aminoácidos/genética , Sítios de Ligação/genética , Compartimento Celular/genética , Núcleo Celular/enzimologia , Centrômero/genética , Centrômero/metabolismo , Centrômero/ultraestrutura , Citoplasma/enzimologia , Citoplasma/genética , Regulação Fúngica da Expressão Gênica/genética , Transporte Proteico/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Schizosaccharomyces/enzimologiaRESUMO
The 32 telomeres in the budding yeast genome cluster in three to seven perinuclear foci. Although individual telomeres and telomeric foci are in constant motion, preferential juxtaposition of some telomeres has been scored. To examine the principles that guide such long-range interactions, we differentially tagged pairs of chromosome ends and developed an automated three-dimensional measuring tool that determines distances between two telomeres. In yeast, all chromosomal ends terminate in TG(1-3) and middle repetitive elements, yet subgroups of telomeres also share extensive homology in subtelomeric coding domains. We find that up to 21 kb of >90% sequence identity does not promote telomere pairing in interphase cells. To test whether unique sequence elements, arm length, or chromosome territories influence juxtaposition, we reciprocally swapped terminal domains or entire chromosomal arms from one chromosome to another. We find that the distal 10 kb of Tel6R promotes interaction with Tel6L, yet only when the two telomeres are present on the same chromosome. By manipulating the length and sequence composition of the right arm of chr 5, we confirm that contact between telomeres on opposite chromatid arms of equal length is favored. These results can be explained by the polarized Rabl arrangement of yeast centromeres and telomeres, which promote to telomere pairing by allowing contact between chromosome arms of equal length in anaphase.
Assuntos
Cromossomos Fúngicos/genética , Troca Genética/genética , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Telômero/metabolismo , Southern Blotting , Eletroforese em Gel de Ágar , Microscopia de Fluorescência , Telômero/genéticaRESUMO
The nucleus is a highly heterogeneous structure, containing various 'landmarks' such as the nuclear envelope and regions of euchromatin or dense heterochromatin. At a morphological level, regions of the genome that are permissive or repressive to gene expression have been associated with these architectural features. However, gene position within the nucleus can be both a cause and a consequence of transcriptional regulation. New results indicate that the spatial distribution of genes within the nucleus contributes to transcriptional control. In some cases, position seems to ensure maximal expression of a gene. In others, it ensures a heritable state of repression or correlates with a developmentally determined program of tissue-specific gene expression. In this review, we highlight mechanistic links between gene position, repression and transcription. Recent findings suggest that architectural features have multiple functions that depend upon organization into dedicated subcompartments enriched for distinct enzymatic machinery.