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

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. HeLa cells were treated with dimethyl sulfate (DMS) and total RNA harvested. Amplicon libraries of endogenous human telomerase RNA (hTR) or transfected overexpressed mutant hTR constructs were prepared by reverse transcription with TGIRT-III and sequences on an Illumina iSeq100 system. Reads were size selected and trimmed with TrimGalore, then mapped to the hTR reference sequence with Bowtie2. Mutations were quantified with DREEM, and normalized mutation frequencies were used as psuedoenergy constraints in RNAstructure. The predicted hTR secondary structures were visualized with VARNA.