Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Bone Marrow Failure Syndromes

Abstract

Edited by Joel GottesfeldNaturally occurring mutations in the ribonucleoprotein reverse transcriptase, telomerase, are associated with the bone marrow failure syndromes dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis. However, the mechanism by which these mutations impact telomerase function remains unknown. Here we present the structure of the human telomerase C-terminal extension (or thumb domain) determined by the method of single-wavelength anomalous diffraction to 2.31 Å resolution. We also used direct telomerase activity and nucleic acid binding assays to explain how naturally occurring mutations within this portion of telomerase contribute to human disease. The single mutations localize within three highly conserved regions of the telomerase thumb domain referred to as motifs E-I (thumb loop and helix), E-II, and E-III (the FVYL pocket, comprising the hydrophobic residues Phe-1012, Val-1025, Tyr-1089, and Leu-1092). Biochemical data show that the mutations associated with dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis disrupt the binding between the protein subunit reverse transcriptase of the telomerase and its nucleic acid substrates leading to loss of telomerase activity and processivity. Collectively our data show that although these mutations do not alter the overall stability or expression of telomerase reverse transcriptase, these rare genetic disorders are associated with an impaired telomerase holoenzyme that is unable to correctly assemble with its nucleic acid substrates, leading to incomplete telomere extension and telomere attrition, which are hallmarks of these diseases.Human telomerase is a ribonucleoprotein reverse transcriptase that replicates the ends of eukaryotic chromosomes (1). The protein subunit, TERT, 2 consists of several domains (TEN, TRBD, RT (fingers and palm), and thumb) organized into a closed, ring configuration, generating a large cavity in the interior of the ring and where the RNA template and the DNA bind during telomere elongation (2-5). The closed configuration of the TERT ring is stabilized by extensive interactions between the thumb and TRBD domains as well as by protein-RNA interactions (3, 6). Several invariable motifs in the interior cavity of the TERT ring coordinate the RNA template and telomeric overhang (RNADNA hybrid) and position the 3Ј end of the DNA for catalysis (2, 3). These include motifs E-I and E-II of the thumb domain, which bind the RNADNA hybrid and stabilize the telomerase elongation complex (2, 7); the primer grip region that guides the DNA at the active site of the enzyme and motifs 2 and BЈ of the fingers and palm domain, respectively, which position the RNA template above the active site of the enzyme for nucleotide binding and selectivity (2, 3).Current evidence shows that the RNA binding domain of telomerase (TRBD) binds the template boundary element (TBE) and the activation domain (CR4/5) of telomerase RNA (8 -12). The TBE is a stem loop located only a few nucleotides upstream of the R...

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

“…This is called the ‘TERT ring’ (Figure 1 ). Other known structures include the TEN domain from Tetrahymena thermophila ( 12 ), the TRBD domain from T. thermophila, Oryzias latipes ( 15 ), and Takifugu rubripes ( 11 , 14 ), and the C-terminal domain of human TERT ( 16 ). Limited structural data on the organization of the telomerase catalytic core restricts the understanding of telomerase function and the rational drug design of anticancer therapeutics ( 17 ).…”

Structural Biology of Telomerase

“…Besides the RBD, the only other domain of vertebrate TERT for which a crystal structure has been reported is of a partial human CTE, missing the amino-terminal region containing the thumb loop, at 2.3 Å resolution (Fig. 3F) (Hoffman et al 2017). The all-helical bundle structure is highly similar to the Tribolium CTE (thumb domain) and revealed the locations of some human mutations associated with the telomerase insufficiency disease dyskeratosis congenita.…”

Current Perspectives of Telomerase Structure and Function in Eukaryotes with Emerging Views on Telomerase in Human Parasites

“…It is composed of α-helices connected by several loops which are involved in stabilization of RNA-DNA duplex [ 42 , 44 ]. The recent crystal structure of human CTE domain identifies three highly conserved regions within it namely motifs E-I, E-II and E-III ( Figure 1 ) [ 45 ]. Mutations within these regions cause several human diseases like dyskeratosis congenital, aplastic anemia and idiopathic pulmonary fibrosis as they are shown to disrupt the binding between telomerase RT and DNA leading to the loss of telomerase activity and processivity [ 45 ].…”