Skip to main content
. Author manuscript; available in PMC: 2015 Apr 24.
Published in final edited form as: Cell. 2014 Apr 24;157(3):651–663. doi: 10.1016/j.cell.2014.03.049

Figure 4.

Figure 4

Increased intron-containing pre-tRNA in CLP1 mutant patient cells and loss of TSEN complex affinity resulting in reduced pre-tRNA cleavage with CLP1-purified complexes. (A) Schematic pre-tRNA with location of the intron, occurring one base 3’ to the anticodon. Primers designed to the individual tRNA homologue exon sequence and the unique consensus intron (red). Amplifications with the 5’-primer and any of the 3’-primers. (B, C) qRT-PCR results showed variable changes in pre-tRNA expression in Affected fibroblasts (i.e. about half were increased and half were decreased in Affected), while most pre-tRNA transcripts from Affected iNeurons were increased (i.e. six of eight were increased). Validation and unchanged pre-tRNAs are shown in Figure S4. (D) Northern blot analysis of Chr14:tRNA19-TyrGTA and Chr19:tRNA10-IleTAT pre-tRNA (intron probes) and tRNA-TyrGTA, tRNA-IleTAT, tRNA-LeuCAA pan mature tRNA (5’-exon probes) transcripts relative to U6 loading control, in duplicate showed similar amounts of pre-tRNA and mature tRNA for all transcripts tested, while Affected iNeurons show a increase in Tyr, Ile, and Leu pre-tRNA transcripts, with a corresponding reduction in processed, mature tRNA. Quantification below, displayed as percent mature or pre-tRNA of total tRNA. (E) Western blot of CLP1-purified complexes showing CLP1 p.R140H, p.R140A, and p.K127A with reduced bound TSEN2 and TSEN34 compared to wt CLP1 (TSEN54 served as a positive control). Quantification of the amount of TSEN2 normalized to FlagHis-tagged protein from four independent replicates (below). (F) Time-course of tRNA endonuclease reactions performed on exogenous radiolabeled Tyrosine (Tyr) pre-tRNA with double affinity purified CLP1-bound complexes. Buffer serves as a negative control and Yeast tRNA endonuclease as a positive control. Reduced endonuclease activity observed with CLP1R140H bound complexes compared with wt, quantified at left. *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001 Students t-test (qPCRs) or one-way ANOVA (pulldown).