TERRA transcripts are bound by a complex array of RNA-binding proteins

Silanes, Isabel López de; d’Alcontres, Martina Stagno; Blasco, Marı́a A.

doi:10.1038/ncomms1032

Cited by 124 publications

(117 citation statements)

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“…1A). These constructs are of identical length as previously used in the other two screens for TERRA interactors (Deng et al 2009;Lopez de Silanes et al 2010). The RNA oligonucleotides were incubated with heavy lysine-and arginine-labeled nuclear extracts (see Methods).…”

Section: Results Quantitative Mass Spectrometry Screenmentioning

confidence: 99%

“…Deng et al (2009) identified two ORC subunits, ORC1 and ORC5, and showed that the ORC complex is recruited to TERRA by TERF2. A separate mass spectrometric analysis mostly identified heterogeneous nuclear ribonucleoproteins (HNRNPs) as TERRA partners and focused on the characterization of HNRNP function in TERRA regulation in mouse fibroblasts (Lopez de Silanes et al 2010). However, there was almost no overlap in the proteins identified by mass spectrometry in these two studies.…”

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confidence: 99%

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Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

et al. 2013

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Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)-based proteomics to obtain a high-confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pulldown experiment and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtained many candidates reported in these two studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knockdown of 10 genes encoding candidate proteins significantly affected total cellular levels of TERRA, and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/ARS2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knockdown of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation.

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Section: Results Quantitative Mass Spectrometry Screenmentioning

confidence: 99%

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confidence: 99%

Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

et al. 2013

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“…8 TERRA acts as a scaffold for the assembly of telomeric proteins involved in telomere maintenance and telomeric heterochromatin formation. 9,10 Importantly, there is also evidence for the presence of TERRA RNA G-quadruplexes in living cells.…”

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confidence: 99%

Mechanical unfolding of long human telomeric RNA (TERRA)

et al. 2013

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We report the first single molecule investigation of TERRA molecules.By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.Telomeres are nucleoprotein structures that protect chromosome ends from being recognized as DNA breaks.1 Mammalian telomere DNA consists of long tandem arrays of double-stranded TTAGGG repeats that are maintained by the telomerase. 2 The 3 0 end of each telomere terminates in a G-rich single-stranded overhang (150-200 nucleotides) that is able to self-fold into G-quadruplexes. The demonstration of the in vivo presence of DNA G-quadruplexes at telomeres suggests that these non-canonical secondary structures have a role in telomere end-protection, and therefore in chromosome stability. 3,4 It has also been found that telomere end-binding proteins control the folding and unfolding of DNA G-quadruplexes in vitro 5 and in vivo. 4Telomeres are transcribed from the subtelomeric regions towards chromosome ends into telomeric repeat-containing RNA (TERRA). 6,7 These non-coding RNA molecules contain subtelomere-derived sequences and an average of 34 GGGUUA repeats at their 3 0 end.8 TERRA acts as a scaffold for the assembly of telomeric proteins involved in telomere maintenance and telomeric heterochromatin formation. 9,10 Importantly, there is also evidence for the presence of TERRA RNA G-quadruplexes in living cells. Here, we report the synthesis and purification of TERRA molecules up to 96nt (twice the largest molecule previously analyzed by NMR), a size similar to the endogenous nuclear TERRA 8 (see ESI †).We have also used optical-tweezers (OT) to study RNA molecules with different numbers of possible quadruplexes ranging from 1 (Q1) to 6 (Q6). CD spectra of the RNA sequence Q4 in 25 mM potassium phosphate pH 7 buffer showed a positive band at 260 nm and a negative band at 240 nm (Fig. 1a), the characteristic signature of parallel-stranded G-quadruplex conformations. This result is in agreement with previously reported observations showing that other telomeric RNA sequences are folded into parallel G-quadruplex structures both in sodium and potassium solutions. 12,13,[16][17][18][19][20] It is also important to mention that no band at 300 nm was observed for this long telomeric RNA as it was reported for 22-mer and 45-mer telomeric RNA in ammonium acetate buffer. 15 On the other hand, the normalized CD spectra of the bimolecular G-quadruplex formed by r(UAGGGUUAGGGU) and Q4 showed a similar shape and amplitude, which indicates that most of the Q4 molecules form four G-quadruplexes (Fig. 1b). 21Thermal stability of Q4 was also measured by recording CD melting curves. To test the influence of the potassium concentration on the thermal stability we performed the experiments in two different potassium-containing buffers (Fig. 1c). Melting temperatures of 65.5 1C and 72.5 1C were obtained in 2...

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“…4,10 In addition, recent studies show that the shelterin component TRF2 and several heterogeneous ribonucleoproteins (hnRNPs) interact with endogenous TERRA in mammalian protein extracts and promote TERRA association to telomeric heterochromatin in vivo. 11,12 While TERRA-associated functions continue to elude researchers, discovery has progressed in TERRA biogenesis. The DNA-dependent RNA polymerase II (RNAPII) uses the C-rich telomeric strand as template to produce TERRA, as demonstrated by substantially decreased TERRA steady-state levels measured in mammalian cells treated with RNAPII inhibitors or in RNAPII-deficient yeast mutants.…”

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confidence: 99%