AGO 2 promotes telomerase activity and interaction between the telomerase components TERT and TERC
2018; Springer Nature; Volume: 20; Issue: 2 Linguagem: Inglês
10.15252/embr.201845969
ISSN1469-3178
AutoresIlaria Laudadio, Francesca Orso, Gianluca Azzalin, Carlo Calabrò, Francesco Berardinelli, Elisa Coluzzi, Silvia Gioiosa, Daniela Taverna, Antonella Sgura, Claudia Carissimi, Valerio Fulci,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle27 December 2018free access Transparent process AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC Ilaria Laudadio Corresponding Author [email protected] orcid.org/0000-0002-0990-3201 Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Francesca Orso Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy Search for more papers by this author Gianluca Azzalin Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Carlo Calabrò Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Francesco Berardinelli Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Elisa Coluzzi Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Silvia Gioiosa CNR, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Bari, Italy Search for more papers by this author Daniela Taverna Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy Search for more papers by this author Antonella Sgura Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Claudia Carissimi Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Valerio Fulci Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Ilaria Laudadio Corresponding Author [email protected] orcid.org/0000-0002-0990-3201 Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Francesca Orso Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy Search for more papers by this author Gianluca Azzalin Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Carlo Calabrò Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Francesco Berardinelli Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Elisa Coluzzi Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Silvia Gioiosa CNR, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Bari, Italy Search for more papers by this author Daniela Taverna Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy Search for more papers by this author Antonella Sgura Department of Science, University of Rome "Roma Tre", Rome, Italy Search for more papers by this author Claudia Carissimi Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Valerio Fulci Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy Search for more papers by this author Author Information Ilaria Laudadio *,1, Francesca Orso2, Gianluca Azzalin1, Carlo Calabrò1, Francesco Berardinelli3, Elisa Coluzzi3, Silvia Gioiosa4, Daniela Taverna2, Antonella Sgura3, Claudia Carissimi1,‡ and Valerio Fulci1,‡ 1Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy 2Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy 3Department of Science, University of Rome "Roma Tre", Rome, Italy 4CNR, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Bari, Italy ‡These authors contributed equally to this work as last authors *Corresponding author. Tel: +39 064457731; E-mail: [email protected] EMBO Rep (2019)20:e45969https://doi.org/10.15252/embr.201845969 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) constitute the core telomerase enzyme that maintains the length of telomeres. Telomere maintenance is affected in a broad range of cancer and degenerative disorders. Taking advantage of gain- and loss-of-function approaches, we show that Argonaute 2 (AGO2) promotes telomerase activity and stimulates the association between TERT and TERC. AGO2 depletion results in shorter telomeres as well as in lower proliferation rates in vitro and in vivo. We also demonstrate that AGO2 interacts with TERC and with a newly identified sRNA (terc-sRNA), arising from the H/ACA box of TERC. Notably, terc-sRNA is sufficient to enhance telomerase activity when overexpressed. Analyses of sRNA-Seq datasets show that terc-sRNA is detected in primary human tissues and increases in tumors as compared to control tissues. Collectively, these data uncover a new layer of complexity in the regulation of telomerase activity by AGO2 and might lay the foundation for new therapeutic targets in tumors and telomere diseases. Synopsis AGO2 promotes telomerase activity and the interaction between TERT and TERC. AGO2 further binds TERC and a small RNA derived from TERC, terc-sRNA, that also stimulates telomerase activity. AGO2 depletion results in telomere shortening, impairment of association between TERT and TERC RNA and decrease of telomerase activity. AGO2 interacts with TERC RNA via CR4/CR5 and TBE domains and binds a small RNA (terc-sRNA), which arises from the 3′end of TERC. Overexpression of terc-sRNA stimulates telomerase activity. Introduction Telomeres, the specialized nucleoprotein complexes present at the ends of linear eukaryotic chromosomes, play essential roles in maintaining genome stability and controlling cell proliferation. The length of human telomeric DNA is maintained by the addition of TTAGGG repeats to telomeres by a ribonucleoprotein (RNP) enzyme, telomerase. The catalytic components of the telomerase RNP are the telomerase reverse transcriptase (TERT) and the H/ACA box telomerase RNA component (TERC), which is the template used for the synthesis of telomeres. TERT and TERC are necessary and sufficient for this reaction 1. However in vivo, the telomerase holoenzyme also includes the accessory proteins dyskerin (DKC1), NOP10, NHP2, and GAR1 2. The formation of the catalytically active telomerase holoenzyme is a highly elaborate and multistep process of RNA maturation, assembly, and trafficking within the nucleus 3. Most human somatic cells express insufficient or undetectable levels of telomerase; thus, telomeres shorten at each cell division, reaching a critical threshold, which triggers cellular senescence. On the contrary, continuously dividing cells such as germ cells, stem cells, and expanding lymphocytes require telomerase activity for maintaining telomere length and surviving. Deficiencies in telomerase activity, maturation, or recruitment to telomeres can lead to human telomeropathies, such as aplastic anemia and dyskeratosis congenita 4. On the other hand, telomerase activity is highly elevated in 85–90% of human cancers and in over 70% of immortalized human cell lines 5, 6, suggesting that the activation of telomerase is crucial for continued cell proliferation. Hence, greater knowledge of telomerase biology and regulation is required to increase our understanding of both human diseases and natural cellular processes. Argonaute (AGO) proteins are small RNA (sRNA)-binding proteins which use the sequence information encoded in the sRNA as a guide to identify complementary target RNAs. Humans have four AGO proteins, AGO1, AGO2, AGO3, and AGO4, sharing about 80% identity in their amino acid sequences 7. Among them, only AGO2 has retained the ability to cleave target RNAs 8. The best known function of AGO proteins is post-transcriptional gene silencing in association with microRNAs. However in the last years, sRNA profiling by next-generation sequencing (sRNA-Seq) allowed the identification of novel classes of sRNAs bound to AGO proteins in mammalian cells, in addition to miRNAs 9-12. Moreover, recent evidences involve AGO proteins in nuclear processes such as transcriptional gene silencing 13, 14, DNA damage 15, chromatin remodeling 9, and splicing 16, 17. These new data strongly suggest that AGO proteins can exert previously unexpected functions. Here, we identify a new function of AGO2 in human telomerase biology. Modulation of AGO2 expression by loss- and gain-of-function approaches results in changes in telomerase activity. Taking advantage of an AGO2 knock-out (KO) human cell line, we demonstrate that AGO2 controls the association between TERT and TERC and this results in telomere shortening, lower proliferation rate in vitro, and tumor growth in vivo. We further discover a new AGO2-bound sRNA, herein referred to as terc-sRNA, which arises from the H/ACA domain of TERC. terc-sRNA is detected not only in human cell lines but also in primary human tissues, and its expression increases in tumor samples as compared to non-tumor tissues. Interestingly, overexpression of terc-sRNA is sufficient to increase telomerase activity. Together, these data show that AGO2 and the newly identified terc-sRNA are novel players in human telomerase regulation. Results Proliferation and tumor growth are affected by AGO2 depletion AGO2 encodes for the only member of the AGO protein family that retains endonucleolytic activity in human cells 8. While its functions in miRNA-mediated post-transcriptional regulation and RNAi have been thoroughly characterized, the roles of AGO2 in the nucleus of human cells, including a role in DNA damage 15, chromatin remodeling 9, and splicing 16, are currently beginning to be elucidated. Because of these unexpected functions of human AGO2, an experimental tool to evaluate AGO2 novel functions in human cell biology is required. Therefore, we took advantage of genome editing to generate inactivating mutations in all three copies of the AGO2 locus present in the human cell line HeLaS3 by using zinc-finger nucleases (AGO2KO cells) 18. Firstly, we investigated how AGO2 depletion might influence cell proliferation and survival. Comparison of growth curves of parental and AGO2KO HeLaS3 cells highlighted that the absence of AGO2 impaired the proliferative capacity of HeLaS3 cells (Fig 1A). Similarly, the results of colony formation assay also showed that clonogenic survival was decreased in AGO2KO cells as compared to HeLaS3 (Fig 1B). We also assessed in vivo proliferation of AGO2KO cells by injecting subcutaneously immunocompromised mice with parental HeLaS3 and AGO2KO cells. In line with in vitro assays, when we measured tumor diameter at different time points (Fig 1C) and tumor weight at 30 days post-injection (Fig 1D), we found that AGO2 depletion inhibits the growth of HeLaS3 xenografts in nude mice. Figure 1. AGO2 depletion in human cells reduces proliferation rate in vitro and tumor growth in vivo Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n = 6 experimental replicates). Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony formation assay (n = 3 experimental replicates). Tumor growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumor diameter at different time points (n = 5 mice for each group). Xenograft tumor weights were measured 30 days post-injection (n = 5 mice for each group). Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.01 (Student's t-test). Download figure Download PowerPoint Next, we profiled the transcriptome of HeLaS3 and AGO2KO cells by microarray. Microarray analysis showed that only 12 genes are differentially expressed in AGO2KO compared to wild-type cells (FDR < 0.01; Appendix Fig S1). Since the endpoint of the microRNA pathway is mRNA degradation, these data suggest that AGO2 is unlikely to control proliferation in human cells via miRNA-mediated regulation of gene expression 19, suggesting the existence of an alternative mechanism. AGO2 interacts with a sRNA arising from TERC locus (terc-sRNA) Several recent papers show that AGO proteins interact with small RNAs other than miRNAs, displaying novel roles in particular in the nuclear compartment 9, 14-17. We recently published that in the nuclei of human cells, AGO2 is bound to sRNAs arising from transcriptional termination sites (TTSa-RNAs). We found that AGO2-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation 18. In HeLaS3 and HCT116 cell lines, nuclear AGO2 interacts with a 23-nt sRNA arising from TTS of the telomerase RNA component TERC (positions 425–447), herein referred to as terc-sRNA (Fig 2A). Based on its association with AGO2, its specific size, and the fact that almost no reads map on TERC RNA outside positions 425–447 (Fig EV1A), we conclude that terc-sRNA is not a mere by-product of TERC degradation, but it is a specific, biologically generated sRNA. We further confirmed that TERC is the precursor of terc-sRNA by stably overexpressing TERC in HeLaS3 cells by lentiviral transduction (Fig 2B). Indeed, we found higher levels of terc-sRNA in the sRNA fraction (< 100 nt) of TERC-overexpressing cells than in control cells (Fig 2C), as assessed by RT–qPCR using specific primers 20 (Appendix Fig 2A–C). Figure 2. AGO2 interacts with a sRNA arising from TERC locus (terc-sRNA) TERC RNA secondary structure (adapted from http://telomerase.asu.edu). terc-sRNA is highlighted in purple HeLaS3 cells were transduced with a lentiviral vector coding for human TERC (HeLaS3_TERC) or for GFP, as a control (HeLaS3_GFP). The relative expression of TERC was measured by RT–qPCR. HPRT1 was used for normalization (n = 3 experimental replicates). The relative expression of terc-sRNA was measured in HeLaS3_TERC and HeLaS3_GFP by RT–qPCR. RNU44 was used for normalization (n = 3 experimental replicates). Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; ***P ≤ 0.01 (Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The newly identified AGO2-bound terc-sRNA is processed in a DICER- and AGO2-independent manner A. Coverage of a sRNA (23 nt) arising from positions 425–447 of mature TERC RNA (terc-sRNA), as assessed by sRNA-Seq of nuclear AGO2-IP from HCT116 and HeLaS3 cells. B. Validation of sRNA-Seq was performed by immunoprecipitating AGO2-bound RNA from HeLaS3 whole-cell extract using a different anti-AGO2 antibody or IgG, as negative control. Immunoprecipitation was verified by Western blot. C. RIP assay was performed from HeLaS3 whole-cell extract using anti-AGO2 antibody or IgG, as negative control. terc-sRNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT–qPCR. 7SK RNA was used for normalization (n = 3 experimental replicates). D–G. terc-sRNA, miR-21, miR-30a, and let7f abundance was assessed in small RNA (< 100 nt) fractions of HCT116 and HCT116 DICEREX5 (D, E) and of HeLaS3 and AGO2KO cells (F, G). RNU44 was used for normalization (n = 3 experimental replicates). Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ns = not significant (Student's t-test). Download figure Download PowerPoint In human cells, impairment of core components of telomerase RNP, TERC and TERT, not only determines short telomeres, but also affects proliferation and colony-forming efficiency, mimicking the phenotype we observed in AGO2KO cells 21, 22. As a consequence, we hypothesized that AGO2 and terc-sRNA might be involved in telomerase-mediated telomere lengthening. AGO2 controls telomere lengthening and enhances telomerase activity To verify this hypothesis, we compared relative telomere length in parental and AGO2KO HeLaS3 by quantitative PCR 23. The ratio of the copy number of telomeric repeats (T) and the copy number of a reference gene was calculated. As a reference, we used multicopy genes, with thousands of copies throughout the genome, similar to telomeres (Alu in Fig 3A and 18S in Appendix Fig S3A), as well as 36B4 (Appendix Fig S3A), which is a four-copy gene in HeLaS3 cells. Regardless of the reference we used, average telomere length in AGO2KO cells was 70% shorter than parental cells. Figure 3. AGO2 controls telomere length and telomerase activity A. Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n = 3 experimental replicates). B. Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA. Scale bar = 5 μm. C. Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift toward short telomeres in AGO2KO cells compared with the parental HeLaS3 cell line. D, E. Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat-inactivated in cell extracts, and in the last lane, no cells were added in lysis buffer. Intensity of the telomerase products (6-bp ladder) was determined by Image Lab Software (Bio-Rad) and normalized with the intensity of the Internal Control (IC; n = 3 experimental replicates). F. AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n = 3 experimental replicates). G. HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n = 3 experimental replicates). Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.01 (Student's t-test). Download figure Download PowerPoint In order to further confirm the observed telomere shortening and analyze single telomere length distributions in AGO2KO and HeLaS3 cells, centromere-calibrated telomeric Q-FISH was performed (Fig 3B). Histograms of telomere length frequencies showed a proportion of short telomeres much higher in AGO2KO cells than in control cells (Fig 3C), and the difference in telomere length was extremely significant and affected both q and p arms (Fig 3C and Appendix Fig S3B). In particular, comparing the percentage of telomeres shorter than three arbitrarily chosen thresholds (i.e., 5, 10, and 15 T/C%), we always found a very significantly higher proportion of eroded telomeres in AGO2KO cells than in HeLaS3 cells (Appendix Fig S3C). Notably, when we measured telomerase activity via Telomerase Repeated Amplification Protocol (TRAP) 24, we observed a decreased telomerase activity in AGO2KO cells as compared to parental cells (Fig 3D and E). Coherently, this phenotype was partially rescued by stable overexpression of FLAG/HA-tagged AGO2 (FH AGO2) in AGO2KO cells (Fig 3F and Appendix Fig S3D). To further confirm that AGO2 expression can modulate telomerase activity, we stably overexpressed FH AGO2 in HeLaS3 parental cells (HeLaS3_FH AGO2; Appendix Fig S3E) and we performed analysis of telomerase activity (Fig 3G). In line with the results obtained in AGO2KO cells, overexpression of AGO2 was sufficient to enhance telomerase activity as compared to control cells. These data show that AGO2 controls telomere elongation capacity and telomerase activity in human cells, pointing out AGO2 as a new player in telomere lengthening. AGO2 affects the association between TERT and TERC We next focused our attention on the mechanism by which AGO2 controls telomerase activity. We hypothesized that AGO2 might directly or indirectly control expression levels of TERT and/or TERC. However, neither TERC RNA levels (Fig 4A) nor TERT protein levels (Fig 4D and E) were affected by AGO2 depletion. Figure 4. AGO2 expression does not affect neither TERC RNA levels and 3′end processing nor TERT protein abundance, but controls association between TERT and TERC A. The relative expression of TERC was measured by RT–qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used for normalization (n = 3 experimental replicates). B. qPCR of 3′-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n = 3 experimental replicates). C. qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n = 3 experimental replicates). D. Whole-cell extract of HeLaS3 and AGO2KO was analyzed by Western blot for the presence of TERT protein. GAPDH was used as loading control. Data are representative of four independent experiments. E. TERT protein level was quantified by Image Lab Software (Bio-Rad). GAPDH was used as loading control (n = 4 experimental replicates). F, G. HeLaS3 and AGO2KO cell extracts (n = 3), as well as AGO2KO_FH AGO2 and AGO2KO_GFP cell extracts (n = 3 experimental replicates), were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT–qPCR. HPRT1 was used for normalization, and enrichment in TERT-RIP as compared to IgG RIP was plotted. H. HeLaS3 whole-cell extract was immunoprecipitated using anti-AGO2 antibody or IgG, as mock IP. Whole-cell lysates (input) and immunoprecipitates were analyzed by Western blot with anti-TERT antibody. As a positive control, the presence in immunoprecipitates of GW182, a known AGO2-interacting protein, was assessed. Data are representative of three independent experiments. Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; ns = not significant (Student's t-test). Download figure Download PowerPoint Furthermore, it has been reported that 3′-extended or polyadenylated isoforms of TERC are post-transcriptionally processed in order to give rise to mature non-polyadenylated 451-nt-long TERC and that this processing is required for telomere maintenance 25, 26. We therefore checked if AGO2 regulates telomerase activity by controlling TERC maturation. We quantified 3′-extended and oligo-adenylated TERC by random hexamer- and oligo d(T)-primed RT–qPCR, respectively, but we did not detect any change in the abundance of immature forms of TERC when AGO2 is ablated (Fig 4B and C). Finally, we aimed to study whether the assembly of the core components of telomerase RNPs, namely TERC and TERT, was affected in AGO2KO cells. By RNA immunoprecipitation (RIP), we looked at endogenous TERC-TERT interaction in both parental and AGO2KO HeLaS3 cells. Notably, we found that enrichment of TERC in TERT-associated RNAs is impaired in AGO2KO cells, as compared to parental HeLaS3 (Fig 4F). As a negative control, we analyzed HOTAIR, which is not enriched in TERT-IP as compared to mock IP (IgG). No variation in HOTAIR enrichment in TERT-IP samples can be detected between AGO2KO and parental HeLaS3. Coherently, re-expression of AGO2 in AGO2KO cells (AGO2KO_FH AGO2) increased association between TERT and TERC as compared to AGO2KO control cells (AGO2KO_GFP; Fig 4G). Importantly, no protein–protein interaction between AGO2 and TERT could be detected by co-immunoprecipitation assay (Fig 4H), suggesting that AGO2 is not part of telomerase RNPs. Overall, our data indicate that AGO2 regulates telomerase activity through the control of the association between TERT and TERC in the assembly of active telomerase RNP. AGO2 is recruited on TERC Since AGO2 is an RNA-binding protein, we checked for interaction between AGO2 and TERC RNA. We firstly performed RIP with an antibody against AGO2 from HeLaS3 cells and we found an enrichment of TERC RNA in AGO2-IP as compared to IgG, as assessed by RT–qPCR (Fig 5A). Association between AGO2 and TERC was further verified by immunoprecipitating FH AGO2 from HeLaS3_FH AGO2 and HeLaS3_GFP cells (as a negative control) with an HA antibody (Fig 5B). Figure 5. AGO2 is bound to TERC RNA, and deletion of TERC regions complementary to terc-sRNA impairs AGO2/TERC interaction HeLaS3 whole-cell extract was immunoprecipitated using anti-AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT–qPCR. 7SK RNA was used for normalization (n = 4 experimental replicates). RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT–qPCR. 7SK RNA was used for normalization (n = 3 experimental replicates). HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313–340 [HeLaS3_TERC (313–340mut)], or TERC delated in position 12–31 [HeLaS3_TERC (Δ 12–31)]. Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC (313–340mut), and HeLaS3_TERC (Δ 12–31) were immunoprecipitated using an anti-AGO2 antibody in order to assess the impact of TERC mutations on AGO2 binding. For each experiment, the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC (313–340mut) and TERC (Δ 12–31) was compared to the association with TERCwt (n = 3 experimental replicates). Data information: Data are expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01 (Student's t-test). Download figure Download PowerPoint We additionally demonstrated the interaction between AGO2 and TERC by taking advantage of HeLaS3 cells stably overexpressing TERC (Fig 2B). Ectopically expressed TERC RNA (ectoTERC) can be discriminated by RT–qPCR from the endogenously expressed one, because it is transcribed from the integrated lentiviral vector as a longer RNA tagged with a specific sequence downstream the 3′end of TERC coding region. Indeed, using specific primers, ectoTERC RNA was detected only in HeLaS3_TERC but not in control HeLaS3 cells (HeLaS3_GFP; Appendix Fig S4A). We immunoprecipitated AGO2, and we found that it is also bound to the ectopically expressed TERC RNA (Appendix Fig S4B). Through base pairing, AGO2-loaded sRNAs recognize complementary RNAs as their targets. terc-sRNA arises from the right arm of the terminal hairpin of TERC, displaying a good complementarity to the left arm of this hairpin. Therefore, we looked for putative target sites of terc-sRNA in TERC RNA by using the RNAhybrid program, which finds the energetically most favorable hybridization sites of a sRNA in a large RNA 27. Surprisingly, the two-best pairing between TERC and terc-sRNA involved positions 313–340 of TERC, in the conserved region 4 (CR4)/CR5 domain of TERC (minimum free energy: −30 kcal/mol) and positions 12–31 of TERC, localized in the template boundary element (TBE) at the 5′end of TERC (minimum free energy: −29.4 kcal/mol; Appendix Fig S4C and D). These data suggest that terc-sRNA not only originates from TERC RNA, but might also target TERC in two different binding sites, guiding interaction between AGO2 and TERC by base pairing. Finally, in order to verify whether the interaction between AGO2 and TERC is mediated by terc-sRNA predicted sites, we destroyed complementarity by mutating TERC sequence in positions 313–340 and by deleting TERC positions 12–31 in the lentiviral vector coding for ectoTERC (TERC (313–340mut) and TERC (Δ 12–31), respectively). HeLaS3 cell were transduced with mutated TERC coding lentiviral particles [HeLaS3_TERC (313–340mut) and HeLaS3_TERC (Δ 12–31)]. The expression levels of ectoTERC were comparable in HeLaS3_TERCwt, HeLaS3_TERC (313–340mut), and HeLaS3_TERC (Δ 12–31; Appendix Fig S4E). We next checked association between AGO2 and ectoTERC, by immunoprecipitating AGO2-associated RNA from lysates of cells expressing the different TERC variants. As shown in Fig 5C, when we compared ectoTERC-AGO2 binding in HeLaS3_TERCwt, HeLaS3_TERC (313–340mut), and HeLaS3_TERC (Δ 12–31), we found that enrichment of ectoTERC in RIP AGO2 samples decreases when terc-sRNA binding sites are mutated. Overall, these data demonstrate that AGO2 binds to TERC. Moreover, positions 313–340 and 12–31 of TERC, displaying base complementarity with terc-sRNA, are required for this interaction, strongly suggesting that terc-sRNA guides AGO2 onto TERC. terc-sRNA biogenesis does not involve DICER or AGO2 By sRNA-Seq, we showed that AGO2 binds to terc-sRNA (Fig EV1A). To confirm this interaction, we immunoprecipitated endogenous AGO2 in HeLaS3 whole-cell extract and we amplified terc-sRNA by RT–qPCR (Fig EV1B and C). In line with high-throughput data, terc-sRNA is enriched in AGO2-IP sample as compared to mock IP, performed using matched immunoglobulin. terc-sRNA derives from a stem-loop structure (Fig 2A), reminiscent of miRNA precursors. We hypothesized that DICER or AGO2, both of which are known to control miRNA processi
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