Physical connectivity mapping by circular permutation of human telomerase RNA reveals new regions critical for activity and processivity

Telomerase is a specialized ribonucleoprotein complex that extends the 3' ends of chromosomes to counteract telomere shortening. However, increased telomerase activity is associated with ~90% of human cancers. The telomerase enzyme minimally requires an RNA (hTR) and a specialized reverse transcriptase protein (TERT) for activity in vitro. Understanding the structurefunction relationships within hTR has important implications for human disease. For the first time, we have tested the physicalconnectivity requirements in the 451-nucleotide hTR RNA using circular permutations, which reposition the 5' and 3' ends. Our extensive in vitro analysis identified three classes of hTR circular permutants with altered function. First, circularly permuting 3' of the template causes specific defects in repeat-addition processivity, revealing that the template recognition element found in ciliates is conserved in human telomerase RNA. Second, seven circular permutations residing within the catalytically important core and CR4/5 domains completely abolish telomerase activity, unveiling mechanistically critical portions of these domains. Third, several circular permutations between the core and CR4/5 significantly increase telomerase activity. Our extensive circular permutation results provide insights into the architecture and coordination of human telomerase RNA and highlight where the RNA could be targeted for the development of antiaging and anticancer therapeutics.