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

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cell

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cells

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

Telomerase Efficiently Elongates Highly Transcribing Telomeres in Human Cancer Cells

RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

PTDC/BIA-MOL/30438/2017 PTDC/MED-OUT/4301/2020 RiboMed 857119 PD/BD/128292/2017 LCF/PR/HP21/52310016 PTDC/BIA-MOL/6624/2020 PTDC/MED-ONC/7864/2020DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.publishersversionpublishe

Saccharomyces cerevisiae Ebs1p is a putative ortholog of human Smg7 and promotes nonsense-mediated mRNA decay

The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive, correlates with Pol II transcription and mRNA expression levels. Changes in Pol II occupancy were detected in a Upf1 deficient (upf1∆) strain, prevalently at genes showing a high Upf1 relative to Pol II association in wild-type. Additionally, an increased Ser2 Pol II signal was detected at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1cells are hypersensitive to the transcription elongation inhibitor 6-azauracil. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1. These data envisage that by operating on the nascent transcript, Upf1 might influence Pol II phosphorylation and transcription

Telomere functions grounding on TERRA firma

Long noncoding telomeric repeat-containing RNAs TERRAs - are transcribed in a regulated manner from telomeres throughout eukaryotes. TERRA molecules consist of chromosome end-specific subtelomeric sequences and telomeric repeats at their 3' ends. Recent work suggests that TERRA sustains several important functions at chromosome ends. TERRA can regulate telomere length through modulation of exonuclease 1 and telomerase, it may promote recruitment of chromatin modifiers to damaged telomeres and thereby enable DNA end-processing, and it may promote telomere protein composition changes during cell cycle progression. Furthermore, telomere transcription regulates chromosome-end mobility within the nucleus. We review how TERRA, by regulated expression and by providing a molecular scaffold for various protein enzymes, can support a large variety of vital functions