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Expression data from from hTR and hTERT knockdown in human CD4 T cells

ABSTRACT: We have found that knockdown of the human telomerase RNA template, hTR, induces Bim-mediated apoptosis independent of telomere length in primary human CD4 T cells, whereas knockdown of the telomerase enzymatic protein, hTERT does not induce apoptosis in the timeframe of our studies. We used microarray analysis to determine any gene changes in human primary CD4 T cells that could provide mechanistic insight into hTR knockdown induced apoptosis. Cells with control shScramble, shTERT, and shTR were used in this experiment

ORGANISM(S): Homo sapiens

PROVIDER: GSE60855 | GEO | 2014/08/28

SECONDARY ACCESSION(S): PRJNA259673

REPOSITORIES: GEO

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Expression data from from hTR and hTERT knockdown in human CD4 T cells

Project description:We have found that knockdown of the human telomerase RNA template, hTR, induces Bim-mediated apoptosis independent of telomere length in primary human CD4 T cells, whereas knockdown of the telomerase enzymatic protein, hTERT does not induce apoptosis in the timeframe of our studies. We used microarray analysis to determine any gene changes in human primary CD4 T cells that could provide mechanistic insight into hTR knockdown induced apoptosis. Cells with control shScramble, shTERT, and shTR were used in this experiment Primary human CD4 T cells were trandsuced in biological triplicates with lentivirus containing shRNAs against a scrambled sequence, hTERT, and hTR 24 hours after CD3 CD28 stimiulation. Cells were cultured in the presence of puromycin to select for cells with shRNAs. shTERT and shTR were compared to shScramble results.

2014-08-28 | E-GEOD-60855 | biostudies-arrayexpress

Next generation sequencing identifies polyadenylated species of hTR

Project description:The paired-end next-generation sequencing of all hTR versions of less than 200 nucleotides in length was used to analyze the 3’ end distribution of hTR associated to Flag-tagged versions of PABPN1, hTERT and Dyskerin. In total, we obtained 2,716,342, 3,152,013, and 3,077,395 hTR-specific reads for the PABPN1, hTERT, and Dyskerin purifications, respectively. We found that the majority of polyadenylated telomerase RNA recovered in the PABPN1, hTERT, and Dyskerin purifications had poly(A) tails starting immediately after the annotated hTR 3’ end. However, a greater proportion of poly(A) tails were found on genome-encoded 3’-extentions in the PABPN1-bound fraction compared to the population of hTR that was copurified with hTERT and DKC1: 15.5% for PABPN1, 6.9% for hTERT, and 6.5% for Dyskerin. Notably, the population of polyadenylated telomerase RNA recovered with PABPN1 was significantly different in terms of poly(A) tail length compared to polyadenylated hTR retrieved in the hTERT- and Dyskerin-bound fractions (P-value=5.551e-16, Kolmogorov-Smirnov test), showing a tendency toward longer poly(A) tails in the PABPN1-bound fraction. The enrichment of telomerase RNA with long poly(A) tails in the PABPN1-bound fraction is consistent with a role of PABPN1 in a maturation pathway that depends on hTR 3’ end polyadenylation. Moreover, our results indicate that a fraction of hTERT and Dyskerin are associated with polyadenylated versions of hTR.

2015-10-21 | GSE74186 | GEO

Differentially expressed genes in CSRP2BP knockdown HTR-8 cells

Project description:CSRP2BP plays an important role in cell cycle progression, apoptosis and mammalian development, CSRP2BP knockdown by specific shRNA (CSRP2BP-shRNA) significantly inhibited the proliferation, migration, and invasion of HTR-8 trophoblast cells, and placental explant outgrowth, while CSRP2BP overexpression did the opposite. To elucidate the mechanisms by which CSRP2BP regulated the function of trophoblast cells, we performed microarray analysis to compare the transcription profiling between the HTR-8-shNC and HTR-8-shCSRP2BP cells

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LARP3, LARP7, and MePCE are Involved in the Early Stage of Human Telomerase RNA Biogenesis

Project description:Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we found that LARP3 and LARP7/MePCE are involved in the early stage and that their binding to hTR is destabilized when the mature hTR is produced. LARP3 plays a negative role in preventing the processing of the 3′-extended long (exL) form and binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Supporting this process, LARP3 at low levels promotes hTR maturation, increases telomerase activity, and elongates telomeres. LARP7 and MePCE knockdown inhibits the conversion of the 3′-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and thus control the production of functional telomerase.

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Differetially expressed genes after hTR overexpression in U2OS cells

Project description:lncRNA is reported to regulate gene transcription in variety of ways. hTR is a lncRNA with 451 nt. It is classical role is serving as template for telomere lengthening. However, it involves in some other biology processes as a lncRNA. To screen for genes regulated by hTR, we performed RNAseq with hTR expressed U2OS cells, using pBabe-U2OS as control.

2019-01-15 | GSE125024 | GEO

Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq

Project description:Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

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Project description:Primary telomerase RNA transcripts are processed into shorter mature forms that assemble into a complex with the catalytic subunit and provide the template for telomerase activity. In diverse fungi telomerase RNA 3â end processing involves a single cleavage reaction by the spliceosome akin to the first step of splicing. Longer forms of human telomerase RNA (hTR) have been reported, but how the mature form of precisely 451 nucleotides is generated is still unknown. We now show that the splicing inhibitor isoginkgetin causes accumulation of long hTR transcripts, but find no evidence for a direct role for splicing in hTR processing. Instead, isoginkgetin mimics the effects of inhibiting the RNA exosome. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly (A) specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and quality control pathways and suggest new treatment options for dyskeratosis congenita caused by mutations in RNA processing factors. We cloned and sequenced 3â ends by RLM-RACE coupled with high-throughput sequencing to gain further insights into hTR processing.

2015-11-25 | E-GEOD-73776 | biostudies-arrayexpress

Maturation of human telomerase RNA component from an extended precursor

Project description:Here, we develop nascent RNAend-Seq, in which we isolate nascent RNA and sequence the 3' ends of RNA precursors. Using a pulse-chase experimental design, we follow extended precursors of the telomerase RNA component (hTR) and show that the mature telomerase RNA derives from these species with slow kinetics compared to other small non-coding RNAs. The human disease causing gene PARN further delays maturation of the hTR precursor in PARN- mutant cancer cell lines. Disruption of the poly(A)polymerase PAPD5 in PARN-mutant cells restores normal processing kinetics and levels of mature hTR. Unexpectedly, neither PARN nor PAPD5 is required for hTR processing. Instead, PAPD5 and PARN set the hTR maturation rate by controlling precursor adenylation.

2019-03-28 | GSE117054 | GEO