Telomeres are transcribed generating long non-coding RNAs known as TERRA. Deciphering the role of TERRA has been one of the unsolved issues of telomere biology in the past decade. This has been, in part, due to lack of knowledge on the TERRA loci, thus preventing functional genetic studies. Here, we describe that long non-coding RNAs with TERRA features are transcribed from the human 20q and Xp subtelomeres. Deletion of the 20q locus by using the CRISPR-Cas9 technology causes a dramatic decrease in TERRA levels, while deletion of the Xp locus does not result in decreased TERRA levels. Strikingly, 20q-TERRA ablation leads to dramatic loss of telomere sequences and the induction of a massive DNA damage response. These findings identify chromosome 20q as a main TERRA locus in human cells and represent the first demonstration in any organism of the essential role of TERRA in the maintenance of telomeres. ; We are indebted to R. Torres and S. Rodriguez for advice in the CRISPR-Cas9 technology and helpful discussions. We thank D. Megías for the quantification of confocal imagesignals and to O. Dominguez for troubleshooting with cloning and sequencing. We thank S. Llanos for the p21 promoter reporter (described in ref. 33). Research in the Blasco lab is funded by the Spanish Ministry of Economy and Competitiveness Project (SAF2013-45111-R), the Madrid Regional Government Project S2010/BMD-2303 (ReCaRe),Fundacio´n Botı´n (Spain) and AXA Research Fund (AXA 2011, Spain). ; Sí
Telomeric RNAs (TERRAs) are UUAGGG repeat-containing RNAs that are transcribed from the subtelomere towards the telomere. The precise genomic origin of TERRA has remained elusive. Using a whole-genome RNA-sequencing approach, we identify novel mouse transcripts arising mainly from the subtelomere of chromosome 18, and to a lesser extend chromosome 9, that resemble TERRA in several key aspects. Those transcripts contain UUAGGG-repeats and are heterogeneous in size, fluctuate in abundance in a TERRA-like manner during the cell cycle, are bound by TERRA RNA-binding proteins and are regulated in a manner similar to TERRA in response to stress and the induction of pluripotency. These transcripts are also found to associate with nearly all chromosome ends and downregulation of the transcripts that originate from chromosome 18 causes a reduction in TERRA abundance. Interestingly, downregulation of either chromosome 18 transcripts or TERRA results in increased number of telomere dysfunction-induced foci, suggesting a protective role at telomeres. ; We are indebted to Stefan Schoeftner and Susana Llanos for reagents and to Manuel Serrano, Maria Elisa Varela and Antonio Maraver for very helpful suggestions and discussion on the manuscript. We thank Diego Megias for confocal image acquisition and to Miguel Angel Grillo, Maria del Carmen Carralero and Juan Cruz Cigudosa for the Spectral Karyotyping (SKY). We thank Luis E. Donate for manuscript preparation. External RNAseq data were generated and analysed by the UW ENCODE group and by the transcriptome group at Cold Spring Harbor Laboratories and the Center for Genomic Regulation (CRG in Barcelona), who are participants in the ENCODE Transcriptome Group. ChIP data of transcription factors binding site were generated and analysed by the laboratories of Michael Snyder at Stanford University and Sherman Weissman at Yale University within the ENCODE Project. Histone marks data belong to the Caltech/ENCODE project in which cell growth, ChIP and Illumina library construction were done in the laboratory of Barbara Wold (California Institute of Technology). Sequencing was done at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology, initial HiSeq data were generated at Illumina Inc., Hawyard, CA. Cell growth and ChIP of histone marks were carried out by Georgi Marinov, Katherine Fisher, Gordon Kwan, Antony Kirilusha, Ali Mortazavi, Gilberto DeSalvo and Brian Williams. Library Construction, Sequencing and Primary Data Handling by Lorianne Schaeffer, Diane Trout, Igor Antoschechkin (California Institute of Technology), Lu Zhang and Gary Schroth (Illumina Inc.). Data processing and submission by Georgi Marinov and Diane Trout. Research in the Blasco laboratory is funded by the Spanish Ministry of Economy and Competitiveness Projects SAF2008-05384 and CSD2007-00017, the Madrid Regional Government Project S2010/BMD-2303 (ReCaRe), the European Union FP7 Project FHEALTH-2010-259749 (EuroBATS), the European Research Council (ERC) Project GA#232854 (TEL STEM CELL), the Preclinical Research Award from Fundacion Lilly (Spain), Fundacion Botin (Spain) and AXA Research Fund. ; Sí
