Saturating variant analysis of TERC redefines the human telomerase reverse transcription mechanism

Genetic variation at the TERC locus, encoding the telomerase RNA component, is associated with human diseases and lifespan, but a comprehensive functional annotation of this critical non-coding RNA is lacking. Here, we performed saturating variant analysis of TERC in cells and unexpectedly redefine the human telomerase catalytic mechanism. Telomerase is understood to precisely reverse transcribe six TERC templating residues to generate GGTTAG repeats, however our screen identified dominant negative effects expected of templating bases inconsistent with this annotation. In vitro and in cells, we found human telomerase uses not six, but eight TERC residues flexibly for reverse transcription, most commonly yielding GGGTTA, but variably up to AGGGTTAG. An evolutionary change in TERC adjacent to the template explains the long-standing misannotation, reversion of which shifts the main templating register in TERC back to the one assumed for decades. Remarkably, this template-adjacent change also yields hyperactive TERC variants that rapidly lengthen telomeres when introduced into cells, including those from patients with genetic telomere diseases. By functional analysis of TERC variation in human cells, our work revises core tenets of telomerase reverse transcription and provides a new mechanistic model to inform the development of telomere-directed therapeutics.