RESUMO
The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATIrDNA" chromosomes), it is dispensable for HAATIrDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATIrDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device.
Assuntos
DNA Ribossômico , Heterocromatina , Interferência de RNA , Translocação Genética , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Rad51 Recombinase/fisiologia , Ribonuclease III/metabolismo , Ribonuclease III/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Complexo Shelterina , Telômero , Proteínas de Ligação a Telômeros/metabolismo , Sequências Repetidas TerminaisRESUMO
Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24(PA) incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24(OR) and UN-24(PA). Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.
Assuntos
Dissulfetos/química , Proteínas Fúngicas/química , Neurospora crassa/enzimologia , Ribonucleotídeo Redutases/química , Alelos , Sequência de Aminoácidos , Domínio Catalítico , Proliferação de Células , Cisteína/química , Cisteína/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neurospora crassa/química , Neurospora crassa/genética , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismoRESUMO
Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to approximately 80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.