Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends (Blackburn and Collins, 2011; Blackburn et al., 2006; Greider et al., 1985; Morin, 1989; Nguyen, 2021). This counterbalances telomere loss caused by incomplete genome replication (Blackburn and Collins, 2011; Greider et al., 1985). Human telomerase has two distinct functional lobes, termed the catalytic core and the Hinge and ACA (H/ACA) box RNP, which are tethered together by telomerase RNA (hTR) (Nguyen et al., 2018). The H/ACA RNP lobe is essential for telomerase biogenesis and hTR accumulation, and it harbours many disease mutation residues (Egan and Collins, 2010, 2012b; Ghanim et al., 2021; Nguyen et al., 2018). H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs (Charette and Gray, 2000). The telomerase catalytic core, on the other hand, is responsible for telomeric DNA extension and telomerase recruitment to telomeres (Egan and Collins, 2010; Nandakumar et al., 2012; Nguyen et al., 2018; Zhong et al., 2012). In mammals, telomerase is recruited to telomeres by the shelterin component TPP1 (Nandakumar et al., 2012; Sexton et al., 2012; Zhong et al., 2012), which forms a heterodimer with another shelterin component POT1 (Xin et al., 2007). After synthesizing approximately 60 telomeric repeats, telomerase is terminated by a CST (CTC1-STN1-TEN1) complex (Feng et al., 2018). In my thesis, I describe the biochemical and structural work aimed at understanding the molecular basis of human telomerase and its interaction with regulatory factors.

In Chapter 2, I reconstruct telomerase complexes with shelterin regulatory factors. This yields the first structures of the human telomerase catalytic core with TPP1 and POT1, resolved at 3.2 and 3.9 Å resolution. The structures elucidate specific interactions between telomerase and TPP1-POT1 that are essential for telomerase recruitment to telomeres and rationalise prior genetic and biochemical findings. Together with mutagenesis studies, the structures reveal an unexpected path of telomeric DNA and provide insights into a DNA anchor site on telomerase, which is important for telomerase processivity.

The same cryo-EM dataset was used to obtain a structure of the telomerase H/ACA RNP at 2.7 Å resolution, which is presented in Chapter 3. This structure reveals unprecedented insights into a region critical for eukaryotic pseudouridylation in canonical H/ACA RNPs. Additionally, we find that many disease-associated residues of the H/ACA RNP proteins form interactions critical for the H/ACA RNP stability.

The cryo-EM structures obtained in Chapter 2 and 3 represent a monomeric configuration of human telomerase with one catalytic core and one H/ACA RNP. In collaboration with S. Balch, who collected a large cryo-EM dataset of human telomerase with various DNA substrates, we resolved the first high-resolution structure of the telomerase dimer. In Chapter 4, preliminary structural and biochemical analyses of the telomerase dimer reveal the unexpected inter-protomer RNA-protein and protein-protein interactions. Moreover, regions of hTR implicated in telomerase dimerization as well as assembly and biogenesis are resolved for the first time.

Finally, to investigate the interplay between telomerase, shelterin and CST, I conducted biochemical and structural analyses of the CST complex in conjunction with TPP1-POT1-TIN2 and telomerase. As presented in Chapter 5, a newly-observed conformation of CST, together with the pull-down experiments, sheds light on CST interaction with shelterin components. Furthermore, preliminary data on the ternary telomerase-TPP1-POT1-TIN2-CST complex provide an invaluable platform for further studying telomerase termination.

Overall, the work presented in my thesis provides a framework for future mechanistic studies on telomerase regulation at telomeres.