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AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC.

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.

SUBMITTER: Laudadio I

PROVIDER: S-EPMC6362350 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC.

Laudadio Ilaria I Orso Francesca F Azzalin Gianluca G Calabrò Carlo C Berardinelli Francesco F Coluzzi Elisa E Gioiosa Silvia S Taverna Daniela D Sgura Antonella A Carissimi Claudia C Fulci Valerio V

EMBO reports 20181227 2

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 < ...[more]

PMID: 30591524

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Project description:ObjectivesYTHDF1 is known as a m6 A reader protein, and many researches of YTHDF1 focused on the regulation of mRNA translation efficiency. However, YTHDF1 is also related to RNA degradation, but how YTHDF1 regulates mRNA degradation is indefinite. Liquid-liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells, and there are few reports focused on the correlation of RNA degradation with LLPS. In this research, we focused on the mechanism of YTHDF1 degraded mRNA through LLPS.Materials and methodsThe CRISPR/Cas9 knock out system was used to establish the YTHDF1 knock out (YTHDF1-KO) cell lines (HEK293 and HeLa) and METTL14 knock out (METTL14-KO) cell line (HEK293). 4SU-TT-seq was used to check the half-life changes of mRNAs. Actinomycin D and qPCR were used to test the half-life changes of individual mRNA. RNA was stained with SYTO RNA-select dye in wild type (WT) and YTHDF1-KO HeLa cell lines. Co-localization of YTHDF1 and AGO2 was identified by immunofluorescence. The interaction domain of YTHDF1 and AGO2 was identified by western blot. Phase separation of YTHDF1 was performed in vitro and in vivo. Fluorescence recovery after photobleaching (FRAP) was performed on droplets as an assessment of their liquidity.ResultsIn this research, we found that deletion of YTHDF1 led to massive RNA patches deposited in cytoplasm. The results of 4SU-TT-seq showed that deletion of YTHDF1 would prolong the half-life of mRNAs. Immunofluorescence data showed that YTHDF1 and AGO2 could co-localize in P-body, and Co-IP results showed that YTHDF1 could interact with AGO2 through YT521-B homology (YTH) domain. We confirmed that YTHDF1 could undergo phase separation in vitro and in vivo, and compared with AGO2, YTHDF1 was more important in P-body formation. The FRAP results showed that liquid AGO2 droplets would convert to gel/solid when YTHDF1 was deleted. As AGO2 plays important roles in miRISCs, we also found that miRNA-mediate mRNA degradation is related to YTHDF1.ConclusionsYTHDF1 recruits AGO2 through the YTH domain. YTHDF1 degrades targeting mRNAs by promoting P-body formation through LLPS. The deletion of YTHDF1 causes the P-body to change from liquid droplets to gel/solid droplets, and form AGO2/RNA patches, resulting in a degradation delay of mRNAs. These findings reveal a previously unrecognized crosstalk between YTHDF1 and AGO2, raising a new sight of mRNA post-transcriptional regulation by YTHDF1.

Project description:The catalytic activity of human Telomerase Reverse Transcriptase (TERT) compensates for the loss of telomere length, eroded during each cell cycle, to ensure a correct division of stem and germinal cells. In human tumors, ectopic TERT reactivation, most frequently due to hotspot mutations in the promoter region (TERTp), i.e. c.1-124 C?>?T, c.1-146 C?>?T, confers a proliferative advantage to neoplastic cells. In gliomas, TERTp mutations (TERTpmut) mainly occur in oligodendroglioma and glioblastoma. We screened, for TERTp hotspot mutations, 301 adult patients with gliomas and identified heterozygous mutations in 239 cases: 94% of oligodendroglioma, 85% of glioblastoma, and 37.5% of diffuse/anaplastic astrocytoma. Besides the recurrent c.1-124 C?>?T and c.1-146 C?>?T, two cases of glioblastoma harbored novel somatic TERTp variants, which consisted of a tandem duplications of 22 nucleotides, i.e. a TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, both located downstream c.1-124 C?>?T and c.1-146 C?>?T. In silico analysis predicted the formation of 119 and 108 new transcription factor's recognition sites for TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, respectively. TERTp duplications (TERTpdup) mainly affected the binding capacity of two transcription factors' families, i.e. the members of the E-twenty-six and the Specificity Protein/Krüppel-Like Factor groups. In fact, these new TERTpdup significantly enhanced the E-twenty-six transcription factors' binding capacity, which is also typically increased by the two c.1-124 C?>?T/c.1-146 C?>?T hotspot TERTpmut. On the other hand, they were distinguished by enhanced affinity for the Krüppel proteins. The luciferase assay confirmed that TERTpdup behaved as gain-of-function mutations causing a 2,3-2,5 fold increase of TERT transcription. The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors, with the identification of new recurrent somatic gain-of-function mutations, occurring in 0.8% of glioblastoma IDH-wildtype.

Dataset Information

AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC.

Publications

AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC.

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