To determine if CLP1R140H-, CLP1R140A- and CLP1K127A-bound protein complexes display altered endonuclease activity we performed an in vitro tRNA cleavage assay using the co-purified protein complexes isolated from the two step elution (Figure S4D) and radiolabeled Tyrosine pre-tRNA with yeast tRNA endonuclease as a positive enzymatic control (Trotta et al., 2006). Consistent with a severe defect in TSEN complex affinity, CLP1R140H-associated protein complexes were unable to cleave pre-tRNA Tyr as compared to wt CLP1-purified complexes at all time points analyzed (Figure 4F). As expected, CLP1R140A and CLP1K127A co-purified complexes displayed more endonuclease activity than CLP1R140H-bound proteins, but less activity than wt (Figure S4F). The data suggest that loss of the TSEN complex from mutant CLP1-associated protein complexes impairs pre-tRNA cleavage activity.
