Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Autor:	Forino NM; Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA, USA., Woo JZ; Department of Microbiology, Harvard Medical School, Boston, MA, USA., Zaug AJ; Department of Biochemistry, University of Colorado, Boulder, CO, USA.; Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA., Jimenez AG; Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA., Edelson E; Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, CA, USA., Cech TR; Department of Biochemistry, University of Colorado, Boulder, CO, USA.; Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA., Rouskin S; Department of Microbiology, Harvard Medical School, Boston, MA, USA., Stone MD; Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Aug 29. Date of Electronic Publication: 2024 Aug 29.
DOI:	10.1101/2023.10.04.560962
Abstrakt:	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 thought to be required for hTERT binding. The proportion of hTR CR4/5 that is folded into the primary functional conformation does not require hTERT expression and the fraction of hTR that assumes a misfolded CR4/5 domain is not refolded by overexpression of its hTERT binding partner. This result suggests a functional role for an RNA folding cofactor other than hTERT during telomerase biogenesis. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in telomerase RNP complexes purified from cells via an epitope tag on hTERT, supporting the hypothesis that only the major CR4/5 conformer is active. 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. Competing Interests: Competing Interests T.R.C. is a scientific advisor for Eikon Therapeutics, Storm Therapeutics, lincSwitch Therapeutics, and Somalogic, Inc. The other authors declare no competing interests.
Databáze:	MEDLINE
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