ABSTRACT
Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2-8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance.
Subject(s)
Nuclear Proteins/genetics , Phosphoproteins/genetics , Protozoan Proteins/genetics , RNA, Fungal/genetics , RNA-Directed DNA Polymerase/genetics , RNA/genetics , Ribonucleoproteins/genetics , Schizosaccharomyces/genetics , Telomerase/genetics , Amino Acid Motifs , Conserved Sequence , Gene Expression , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Protozoan Proteins/metabolism , RNA/metabolism , RNA Stability , RNA, Fungal/metabolism , RNA-Directed DNA Polymerase/metabolism , Ribonucleoproteins/metabolism , Schizosaccharomyces/metabolism , Telomerase/metabolism , Telomere/chemistry , Telomere/ultrastructure , Tetrahymena thermophila/genetics , Tetrahymena thermophila/metabolismABSTRACT
Shelterin, the telomeric protein complex, plays a crucial role in telomere homeostasis. In fission yeast, telomerase is recruited to chromosome ends by the shelterin component Tpz1 and its binding partner Ccq1, where telomerase binds to the 3' overhang to add telomeric repeats. Recruitment is initiated by the interaction of Ccq1 with the telomerase subunit Est1. However, how telomerase is released following elongation remains to be established. Here, we show that Ccq1 also has a role in the suppression of telomere elongation, when coupled with the Clr4 histone H3 methyl-transferase complex and the Clr3 histone deacetylase and nucleosome remodelling complex, SHREC. We have dissected the functions of Ccq1 by establishing a Ccq1-Est1 fusion system, which bypasses the telomerase recruitment step. We demonstrate that Ccq1 forms two distinct complexes for positive and negative telomerase regulation, with Est1 and Clr3 respectively. The negative form of Ccq1 promotes dissociation of Ccq1-telomerase from Tpz1, thereby restricting local telomerase activity. The Clr4 complex also has a negative regulation activity with Ccq1, independently of SHREC. Thus, we propose a model in which Ccq1-Est1 recruits telomerase to mediate telomere extension, whilst elongated telomeric DNA recruits Ccq1 with the chromatin-remodelling complexes, which in turn releases telomerase from the telomere.
Subject(s)
Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Telomerase/metabolism , Telomere Homeostasis , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins , Histone-Lysine N-Methyltransferase , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Domains , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Telomerase/chemistry , Telomerase/geneticsABSTRACT
During meiotic prophase, chromosome arrangement and oscillation promote the pairing of homologous chromosomes for meiotic recombination. This dramatic movement involves clustering of telomeres at the nuclear membrane to form the so-called telomere bouquet. In fission yeast, the telomere bouquet is formed near the spindle pole body (SPB), which is the microtubule organising centre, functionally equivalent to the metazoan centrosome. Disruption of bouquet configuration impedes homologous chromosome pairing, meiotic recombination and spindle formation. Here, we demonstrate that the bouquet is maintained throughout meiotic prophase and promotes timely prophase exit in fission yeast. Persistent DNA damages, induced during meiotic recombination, activate the Rad3 and Chk1 DNA damage checkpoint kinases and extend the bouquet stage beyond the chromosome oscillation period. The auxin-inducible degron system demonstrated that premature termination of the bouquet stage leads to severe extension of prophase and consequently spindle formation defects. However, this delayed exit from meiotic prophase was not caused by residual DNA damage. Rather, loss of chromosome contact with the SPB caused delayed accumulation of CDK1-cyclin B at the SPB, which correlated with impaired SPB separation. In the absence of the bouquet, CDK1-cyclin B localised near the telomeres but not at the SPB at the later stage of meiotic prophase. Thus, bouquet configuration is maintained throughout meiotic prophase, by which this spatial organisation may facilitate local and timely activation of CDK1 near the SPB. Our findings illustrate that chromosome contact with the nuclear membrane synchronises meiotic progression of the nucleoplasmic chromosomes with that of the cytoplasmic SPB.
