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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE245536 | GEO | 2023/10/20

REPOSITORIES: GEO

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Human telomerase requires TCAB1 for catalytic function and for shaping RNA component conformation

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2018-06-24 | GSE97486 | 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

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

2014-08-28 | GSE60855 | GEO

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.

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miRNA Expression in Senescent and Immortalized BJ fibroblasts

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2010-06-05 | GSE22134 | GEO

Homo sapiens immortalized MSCs: KP vs. 3A6

2011-08-24 | E-GEOD-23936 | biostudies-arrayexpress

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2018-02-09 | GSE104672 | GEO

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