The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organism

FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops

© The Author(s) 2019. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.Research in the Azzalin laboratory was supported by the Swiss National Science Foundation (31003A_160338), the European Molecular Biology Organization (IG3576) and the Fundação para a Ciência e a Tecnologia (IF/01269/2015; PTDC/MED-ONC/28282/2017; PTDC/BIA-MOL/29352/2017). R.P. was supported by a Swiss National Science Foundation Doc.Mobility fellowship (P1EZP3-168771). Research in the Deans laboratory was supported by the Cancer Council of Victoria, Australian National Health and Medical Research Council (APP1139099), Buxton trust and the Victorian Government’s OIS Program. A.J.D is a Victorian Cancer Agency fellow. Publication costs were supported by UID/BIM/50005/2019, project funded by the Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado.info:eu-repo/semantics/publishedVersio

The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organisms

3D Synthetic Microstructures Fabricated by Two-Photon Polymerization Promote Homogeneous Expression of NANOG and ESRRB in Mouse Embryonic Stem Cells

The development of in vitro models, which accurately recapitulate early embryonic development, is one of the fundamental challenges in stem cell research. Most of the currently employed approaches involve the culture of embryonic stem cells (ESCs) on 2D surfaces. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic 3D microenvironments, as in an early embryo. In this paper, novel 3D synthetic microstructure arrays, fabricated by two-photon polymerization photolithography, are utilized to mimic tissue-specific architecture, enabling cell-to-matrix interaction and cell-to-cell communication in vitro. Mouse ESCs (mESCs) are able to grow and proliferate on these structures and maintain their pluripotent state. Furthermore, the 3D microstructure arrays are integrated into a microscopy slide allowing the evaluation of the expression of key pluripotency factors at the single cell level. Comparing 2D and 3D surfaces, mESCs grown in serum + leukemia inhibitory factor on 3D microstructures exhibit a stronger signal intensity of three pluripotency markers-homeobox protein NANOG, octamer-binding transcription factor 4, and estrogen-related receptor beta (ESRRB)-and more homogenous expression of NANOG and ESRRB, than cells cultivated in 2i medium, demonstrating that 3D microstructures capture naive pluripotency in vitro. Thus, the slide affords a novel and unique tool to model and study early development.ISSN:2196-735

Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

Clericuzio-type poikiloderma with neutropenia (PN) is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA) posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis

RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells

A fraction of cancer cells maintain telomeres through the telomerase-independent, ‘Alternative Lengthening of Telomeres’ (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA–DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA–DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.ISSN:2041-172

Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels

The telomeric transcriptome comprises multiple long non-coding RNAs generated by transcription of linear chromosome ends. In a screening performed in Schizosaccharomyces pombe, we identified factors modulating the cellular levels of the telomeric transcriptome. Among these factors, Cay1 is the fission yeast member of the conserved family of Cactins, uncharacterized proteins crucial for cell growth and survival. In cay1∆ mutants, the cellular levels of the telomeric factor Rap1 are drastically diminished due to defects in rap1+ pre-mRNA splicing and Rap1 protein stability. cay1∆ cells accumulate histone H3 acetylated at lysine 9 at telomeres, which become transcriptionally desilenced, are over-elongated by telomerase and cause chromosomal aberrations in the cold. Overexpressing Rap1 in cay1+ deleted cells significantly reverts all telomeric defects. Additionally, cay1∆ mutants accumulate unprocessed Tf2 retrotransposon RNA through Rap1-independent mechanisms. Thus, Cay1 plays crucial roles in cells by ultimately harmonizing expression of transcripts originating from seemingly unrelated genomic loci

TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe

ISSN:1469-221XISSN:1469-317

Induced neural phase precession through exogenous electric fields

Abstract The gradual shifting of preferred neural spiking relative to local field potentials (LFPs), known as phase precession, plays a prominent role in neural coding. Correlations between the phase precession and behavior have been observed throughout various brain regions. As such, phase precession is suggested to be a global neural mechanism that promotes local neuroplasticity. However, causal evidence and neuroplastic mechanisms of phase precession are lacking so far. Here we show a causal link between LFP dynamics and phase precession. In three experiments, we modulated LFPs in humans, a non-human primate, and computational models using alternating current stimulation. We show that continuous stimulation of motor cortex oscillations in humans lead to a gradual phase shift of maximal corticospinal excitability by ~90°. Further, exogenous alternating current stimulation induced phase precession in a subset of entrained neurons (~30%) in the non-human primate. Multiscale modeling of realistic neural circuits suggests that alternating current stimulation-induced phase precession is driven by NMDA-mediated synaptic plasticity. Altogether, the three experiments provide mechanistic and causal evidence for phase precession as a global neocortical process. Alternating current-induced phase precession and consequently synaptic plasticity is crucial for the development of novel therapeutic neuromodulation methods

Non-targeted metabolomic approach reveals two distinct types of metabolic responses to telomerase dysfunction in S. cerevisiae

Introduction The alternative lengthening of telomeres (ALT) mechanism was first observed in the model organism S. cerevisiae. Interestingly, this mechanism is necessary for the viability of some tumor cells. Unfortunately, its molecular underpinnings are not yet completely understood. Objective Here, we combine carefully designed non-targeted mass spectrometry-based metabolomics experiments with a bioinformatics approach to characterize the ALT positive phenotype observed in yeast at the metabolomics level. Methods We profiled the metabolome using mass spectrometry in yeast strains that have lost telomerase expression, as well as that in pre-senescence and the rescued states. To dissect unwanted technical variation from biologically relevant variation between these states, we used a two-step normalization strategy, i.e., first, an empirical Bayesian framework; and next, we corrected for second-order technical effects. Results Our results show that ALT-positive yeast strains present two different types of metabolic responses to the genetically-induced telomerase dysfunction: (i) systemic and (ii) specific. The key-difference between these responses is that the systemic response lasts even after the yeast strains have been genetically rescued, while the specific response does not. Interestingly, these metabolic changes can be associated with generic stress responses (e.g., DNA damage) as well as specific responses like accelerated aging of early telomerase-inactivation. Conclusions A mass spectrometry-based metabolomics approach reveals two distinct types of metabolomics response to telomerase dysfunction in yeast. By identifying these changes in protein (e.g., ARG7, and ARG1), and metabolite (e.g., dATP, and dDTP) amounts, we complement the available information on ALT at the genome-wide level.ISSN:1573-3882ISSN:1573-389