Telomere deprotection occurs during tumorigenesis and aging upon telomere shortening or loss of the telomeric shelterin component TRF2. Deprotected telomeres undergo changes in chromatin structure and elicit a DNA damage response (DDR) that leads to cellular senescence. The telomeric long noncoding RNA TERRA has been implicated in modulating the structure and processing of deprotected telomeres. Here, we characterize the human TERRA transcriptome at normal and TRF2-depleted telomeres and demonstrate that TERRA upregulation is occurring upon depletion of TRF2 at all transcribed telomeres. TRF2 represses TERRA transcription through its homodimerization domain, which was previously shown to induce chromatin compaction and to prevent the early steps of DDR activation. We show that TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and end-to-end fusions. Altogether our data elucidate the TERRA landscape and defines critical roles for this RNA in the telomeric DNA damage response.
Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5 ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A) ؊ TERRA, but not poly(A) ؉ TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A) ؊ and poly(A) ؉ TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G 1 . Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.The ends of eukaryotic chromosomes, known as telomeres, play crucial roles as guardians of genome stability and tumor suppressors (1, 17). Human telomeres consist of tandem 5Ј-T TAGGG-3Ј/5Ј-CCCTAA-3Ј repeats. The guanine-rich strand is directed in a 5Ј-to-3Ј manner toward the chromosome end and extends beyond its complement strand to form a 3Ј overhang. Telomeric DNA is associated with the six-polypeptide shelterin complex (18), and shelterin components have been demonstrated to protect the ends of chromosomes from being recognized as sites of DNA damage. Human telomeres vary in length between 5 and 15 kb, and they shorten during the course of life, due to the so-called DNA end replication problem and nucleolytic processing (19,46,47).Telomere shortening is counteracted by telomerase, a ribonucleoprotein enzyme that can extend the 3Ј ends of chromosomes. Telomerase works as a reverse transcriptase, using a small region of its tightly associated RNA moiety as a template for the synthesis of telomeric repeats at chromosome ends (22,23,29). The progressive erosion of telomeres and the activation of telomerase play key roles in chromosomal stability, cellular immortalization, and tumor progression (7, 26). Short telomeres elicit a DNA damage signal cascade that leads to growth arrest and replicative senescence (16). Reactivation of telomerase in human cancer cells allows overcoming of the senescence barrier and is a key requisite of cancer cells to attain unlimited proliferation potential.Despite the fact that telomeres are heterochromatic structures, eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA) (4,31,40). TERRA is an integral part of telo...
Oxidative lesions represent the most abundant DNA lesions within the cell. In the present study, we investigated the impact of the oxidative lesions 8-oxoguanine, thymine glycol and 5-hydroxyuracil on RNA polymerase II (RNA pol II) transcription using a well-defined in vitro transcription system. We found that in a purified, reconstituted transcription system, these lesions block elongation by RNA pol II to different extents, depending on the type of lesion. Suggesting the presence of a bypass activity, the block to elongation is alleviated when transcription is carried out in HeLa cell nuclear extracts. By purifying this activity, we discovered that TFIIF could promote elongation through a thymine glycol lesion. The elongation factors Elongin and CSB, but not TFIIS, can also stimulate bypass of thymine glycol lesions, whereas Elongin, CSB and TFIIS can all enhance bypass of an 8-oxoguanine lesion. By increasing the efficiency with which RNA pol II reads through oxidative lesions, elongation factors can contribute to transcriptional mutagenesis, an activity that could have implications for the generation or progression of human diseases.
Telomeres protect chromosome ends from being recognized as sites of DNA damage. Upon telomere shortening or telomere uncapping induced by loss of telomeric repeat-binding factor 2 (TRF2), telomeres elicit a DNA-damage response leading to cellular senescence. Here, we show that following TRF2 depletion, the levels of the long noncoding RNA TERRA increase and LSD1, which binds TERRA, is recruited to telomeres. At uncapped telomeres, LSD1 associates with MRE11, one of the nucleases implicated in the processing of 3' telomeric G overhangs, and we show that LSD1 is required for efficient removal of these structures. The LSD1-MRE11 interaction is reinforced in vivo following TERRA upregulation in TRF2-deficient cells and in vitro by TERRA-mimicking RNA oligonucleotides. Furthermore, LSD1 enhances the nuclease activity of MRE11 in vitro. Our data indicate that recruitment of LSD1 to deprotected telomeres requires MRE11 and is promoted by TERRA. LSD1 stimulates MRE11 catalytic activity and nucleolytic processing of uncapped telomeres.
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