Next we analyzed whether the alterations in pre-tRNA levels were due to impaired TSEN-CLP1 complex formation resulting from the CLP1 p.R140H mutation. Using an established assay (Figure S4D) (Paushkin et al., 2004), we found reduced amounts of the active site containing TSEN complex proteins, TSEN2 and TSEN34 co-purified with double-affinity tagged CLP1R140H, compared to complexes purified with wt CLP1 or TSEN54 (Figure 4E, S4E). Additionally, we generated two control mutants, CLP1R140A and CLP1K127A, and observed a less severe reduction of TSEN2 and TSEN34 bound compared to CLP1R140H, indicating the patient substitution p.140H is particularly damaging to TSEN complex formation (Figure 4E, S4E) (Hanada et al., 2013). We conclude that mutant CLP1 has altered affinity for the TSEN complex.
