Characterization of TPP1 OB-fold variants extend TEL patch functional domain. (A) In silico model of the TPP1 OB-fold crystal structure (Protein Data Bank accession number 2I46)¹⁶  using UCSF CHIMERA, an extensible molecular modeling software. Previously described TPP1-TEL patch residues are denoted in black,¹²  and the patient variants in BMF patients identified to date are denoted in red. OB-fold ribbon structure is depicted in blue, and surface hydrophobicity preset is shown with amino acid hydrophobicity in the Kyte-Doolittle scale with colors ranging from dodger blue for the most hydrophilic to white at 0.0 to orange for the most hydrophobic. NOB refers to the N terminus of the OB-fold domain that is recently described in telomerase binding.¹⁴  (B-C) Immunoblotting of FLAG pull-down (PD) complexes from nuclear extracts of HEK293 cells that are treated with TPP1 3′ untranslated region shRNA and expressing shRNA-resistant FLAG-TPP1 variants along with TERT and POT1. IN refers to 15% input. Note the reduction in TERT signal pulled by the TPP1 OB-fold variants compared with input and WT. (D-E) HeLa cells expressing FLAG-tagged WT TPP1 were analyzed for TPP1-associated endogenous telomerase activity by immunoprecipitation and subsequent telomerase repeated amplification (TRAP) analysis. Results from TRAP assay were normalized based on the amount of eluted TPP1 protein. (F-G) Double immunofluorescence was used to detect the FLAG-TPP1 proteins (green) and coilin (a Cajal body marker, red) in HeLa cells. Yellow (merged green + red) spots (indicated by arrows) show colocalization of TPP1 with coilin, which was quantified by counting cells from different fields of view (n = 2). (H-I) Endogenous TPP1 colocalization with coilin in control and patient (TPP1 p.L95Q) B lymphoblastoid cells. (J) Relative levels of telomerase activity in patient and age-matched control B lymphoblastoid cells at passage 5 were determined by TRAP assay.