RESUMO
Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional shelterin complex associates with telomeres to coordinate multiple telomere activities, including telomere elongation by telomerase. Similar to the human shelterin, fission yeast shelterin is composed of telomeric sequence-specific double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. Here, we report the crystal structure of the fission yeast Tpz1475-508-Poz1-Rap1467-496 complex that provides the structural basis for shelterin bridge assembly. Biochemical analyses reveal that shelterin bridge assembly is a hierarchical process in which Tpz1 binding to Poz1 elicits structural changes in Poz1, allosterically promoting Rap1 binding to Poz1. Perturbation of the cooperative Tpz1-Poz1-Rap1 assembly through mutation of the "conformational trigger" in Poz1 leads to unregulated telomere lengthening. Furthermore, we find that the human shelterin counterparts TPP1-TIN2-TRF2 also assemble hierarchically, indicating cooperativity as a conserved driving force for shelterin assembly.
Assuntos
Proteínas de Transporte/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Proteínas de Ligação a Telômeros/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA , Humanos , Estrutura Quaternária de Proteína , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Schizosaccharomyces pombe/genética , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Cromossomos/genética , DNA/genética , DNA de Cadeia Simples/genética , Humanos , Cinética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Mutação , Schizosaccharomyces/genética , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/ultraestruturaRESUMO
Long non-coding RNAs can often fold into different conformations. Telomerase RNA, an essential component of the telomerase ribonucleoprotein (RNP) enzyme, must fold into a defined structure to fulfill its function with the protein catalytic subunit (TERT) and other accessory factors. However, the mechanism by which the correct folding of telomerase RNA is warranted in a cell is still unknown. Here we show that La-related protein Pof8 specifically recognizes the conserved pseudoknot region of telomerase RNA and instructs the binding of the Lsm2-8 complex to its mature 3' end, thus selectively protecting the correctly folded RNA from exonucleolytic degradation. In the absence of Pof8, TERT assembles with misfolded RNA and produces little telomerase activity. Therefore, Pof8 plays a key role in telomerase RNA folding quality control, ensuring that TERT only assembles with functional telomerase RNA to form active telomerase. Our finding reveals a mechanism for non-coding RNA folding quality control.
Assuntos
Dobramento de RNA , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Telomerase/biossíntese , Domínio Catalítico , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Controle de Qualidade , RNA/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Fúngico , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Motivos de Ligação ao RNA , Ribonucleoproteínas Nucleares Pequenas/química , Schizosaccharomyces/química , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/química , Telomerase/genética , Telomerase/metabolismoRESUMO
Tightly controlled recruitment of telomerase, a low-abundance enzyme, to telomeres is essential for regulated telomere synthesis. Recent studies in human cells revealed that a patch of amino acids in the shelterin component TPP1, called the TEL-patch, is essential for recruiting telomerase to telomeres. However, how TEL-patch-telomerase interaction integrates into the overall orchestration of telomerase regulation at telomeres is unclear. In fission yeast, Tel1(ATM)/Rad3(ATR)-mediated phosphorylation of shelterin component Ccq1 during late S phase is involved in telomerase recruitment through promoting the binding of Ccq1 to a telomerase accessory protein Est1. Here, we identify the TEL-patch in Tpz1(TPP1), mutations of which lead to decreased telomeric association of telomerase, similar to the phosphorylation-defective Ccq1. Furthermore, we find that telomerase action at telomeres requires formation and resolution of an intermediate state, in which the cell cycle-dependent Ccq1-Est1 interaction is coupled to the TEL-patch-Trt1 interaction, to achieve temporally regulated telomerase elongation of telomeres.
Assuntos
Nucleotidases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Mutação , Nucleotidases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência , Complexo Shelterina , Telomerase/genética , Proteínas de Ligação a TelômerosRESUMO
Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between shelterin connectivity and tumorigenicity.