![Figure 1. Mutations in TRNT1 in SIFD patients are loss-of-function alleles. (A) Yeast cca1-1 cells were transformed with centromeric plasmids encoding yeast CCA1, human TRNT1, or an empty vector control (EV) and grown at permissive (22°C) or nonpermissive (37°C) temperatures overnight in selective media. The optical density of the sample measured at a wavelength of 600 nm (OD600) value for the empty vector was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a CCA1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 5). The ability of human TRNT1 to rescue growth at the nonpermissive temperature was not statistically different from CCA1 as determined by Student paired t test. (B) Patient TRNT1 mutants partially rescue growth impairment of cca1-1 cells. cca1-1 cells were transformed with EV, wild-type TRNT1, or TRNT1 mutants (p.T154I, p.L166S, p.R190I, p.M158V, p.I223T, and p.I326T) and grown as in (a). The OD600 value for the EV control was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a TRNT1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 3; Student paired t test *P ≤ .05). (C) Complemented cca1∆ strains that contain only the TRNT-CEN-TRP1 plasmid were isolated from SC-trp + 5-FOA plates (supplemental Figure 15B). Ten-fold serial dilutions of 106 cells of the indicated genotypes were spotted on YEP + 2% dextrose or 3% glycerol plates at the indicated temperatures. Wild-type and most TRNT1 mutants have growth defects and temperature sensitivity, with the exception of TRNT1-p.K416E and TRNT-p.I326T, which are indistinguishable from CCA1 cells. (D) Two different concentrations of purified TRNT1-FLAG protein with patient-specific mutations were incubated with [α32P]-adenosine triphosphate and tRNAAsp lacking the terminal CCA sequence at 37°C for 5 minutes as described in the supplemental Methods and the reactions were terminated by addition of loading dye and resolved on a denaturing 5% polyacrylamide/8M urea gel, dried, and exposed to a phosphoimager screen. Other than the T154I variant, all of the mutants have negligible activity in this assay.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/18/10.1182_blood-2014-08-591370/4/2867f1.jpeg?Expires=1736039582&Signature=xI1oQ28EJB3ivKyoRyZMiXyT1I7MtVxdww4ywAYVBX6Zz8JXdXhYwWk38NpTB8yIFLilDxOI3QiiY1bsuNAIR5aquiHjQ5RtQ7yitY8thplsvxlQEfp5duq156r3PcBZixKBjn1kmPzgUopbLfBxntDAu26HV4c~Rgyruy2hwhJbp-s5OT65IpWK2egIeyI0G1roSlgLkLjVTzY3QNc3uI-BDgIe5VCdQmGY5a7~ae1iV3GjoLTJLEoSgcAV~T5exxMFfa41UoCt2PHeWQVlYzcX41utACajFkBHqYR1Pn4GLQQNV4lsk6hKIMTbFxtJXDX1WYc8H3TUm7t3BLr~Bg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mutations in TRNT1 in SIFD patients are loss-of-function alleles. (A) Yeast cca1-1 cells were transformed with centromeric plasmids encoding yeast CCA1, human TRNT1, or an empty vector control (EV) and grown at permissive (22°C) or nonpermissive (37°C) temperatures overnight in selective media. The optical density of the sample measured at a wavelength of 600 nm (OD600) value for the empty vector was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a CCA1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 5). The ability of human TRNT1 to rescue growth at the nonpermissive temperature was not statistically different from CCA1 as determined by Student paired t test. (B) Patient TRNT1 mutants partially rescue growth impairment of cca1-1 cells. cca1-1 cells were transformed with EV, wild-type TRNT1, or TRNT1 mutants (p.T154I, p.L166S, p.R190I, p.M158V, p.I223T, and p.I326T) and grown as in (a). The OD600 value for the EV control was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a TRNT1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 3; Student paired t test *P ≤ .05). (C) Complemented cca1∆ strains that contain only the TRNT-CEN-TRP1 plasmid were isolated from SC-trp + 5-FOA plates (supplemental Figure 15B). Ten-fold serial dilutions of 106 cells of the indicated genotypes were spotted on YEP + 2% dextrose or 3% glycerol plates at the indicated temperatures. Wild-type and most TRNT1 mutants have growth defects and temperature sensitivity, with the exception of TRNT1-p.K416E and TRNT-p.I326T, which are indistinguishable from CCA1 cells. (D) Two different concentrations of purified TRNT1-FLAG protein with patient-specific mutations were incubated with [α32P]-adenosine triphosphate and tRNAAsp lacking the terminal CCA sequence at 37°C for 5 minutes as described in the supplemental Methods and the reactions were terminated by addition of loading dye and resolved on a denaturing 5% polyacrylamide/8M urea gel, dried, and exposed to a phosphoimager screen. Other than the T154I variant, all of the mutants have negligible activity in this assay.
