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S of the thio-modification of cytosolic tRNAs and growth under stress S of the thio-modification of cytosolic tRNAs and growth under stress conditions. Yeast uba4 mutants that were transformed with plasmids encoding AtCnx5, ScUba4, and the AtCnx5 mutant (Met1 la335) lacking the Lys C-terminal RHD domain (AtCnx5-w/oRHD) were analyzed for the thio-modification of the cytosolic tRNAs Sc-cyK (against cytosolic tRNAUUU) (left) and Sc-cyE Glu (against cytosolic tRNAUUC) (right) using a gel retardation assay (A). Yeast cells that contained a vector alone ( ) were also analyzed. The error bars represent the standard deviation. In B, the uba4 mutant cells that expressed AtCnx5, ScUba4-w/oRHD, ScUba4, or AtCnx5-w/oRHD were examined for their ability to grow under stress Title Loaded From File conditions in the absence (top) or presence of 2 nM rapamycin (middle) or 400 M diamide (bottom) using spot dilution assays. Representative data obtained from three repeated experiments are shown.involved in the thio-modification of tRNAs has not yet been investigated. To examine this question, we analyzed a T-DNA insertion mutant of the AtCNX5 gene (hereafter called cnx5-1) that originated from the FLAGdb/FST collection line FLAG_116G03 (Fig. 1B). When the progeny of the heterozygous cnx5-1/ plant were grown on sucrose-containing medium, a proportion of the seedlings exhibited a striking dwarf phenotype with severe growth defects (19.0 ; 205 dwarf plants of 1078 seedlings) (Fig. 1C). They had slightly green and morphologically aberrant leaves and did not grow well. The seedlings did not produce a stalk or flower, and they were therefore sterile. Genomic PCR genotyping revealed that all of the dwarf individuals were homozygous cnx5-1 mutants. However, the heterozygous cnx5-1/ mutants did not show any growth defects compared with the homozygous CNX5( / ) plants. The observed proportion of homozygous dwarf plants was clearly less than the expected value of 25 for normal inheritance, which suggests that the homozygotes have reduced viability at an early developmental stage, such as seed formation or maturation. The 3-week-old homozygous cnx5-1 mutants and CNX5 ( / ) plants were collected separately, and their total low molecular weight RNA was subjected to a gel retardation assay to analyze the accumulation of thio-modified cytosolic tRNA (Fig. 1D). In this Northern assay, the thio-modification status of any tRNA can be assessed through the retardation of the electrophoretic migration of its band, which is detected using a specific probe for each tRNA in an [(N-acryloylamino)phenyl]mercuric chlorideAUGUST 31, 2012 ?VOLUME 287 ?NUMBERcontaining gel. A significant fraction of the tRNAs from the wild-type plants showed retardation when specific probes corLys responding to either A. thaliana cytosolic tRNAUUU (At-cyK) Glu or A. thaliana cytosolic tRNAUUC (At-cyE) were used for detection; both of these tRNAs are encoded in the nuclear genome of Arabidopsis. These data indicate that a significant proportion of these specific cytosolic tRNAs are thio-modified in Arabidopsis. In contrast, no such retardation was observed when tRNAs from the homozygous cnx5-1 plants were analyzed. The impairment in the thio-modification of cytosolic tRNAs in the homozygous cnx5-1 mutant clearly indicates the critical contribution of AtCnx5 to the thio-modification of At-cyK and At-cyE tRNAs. This conclusion was further confirmed by similar analyses using a subsequently identified additional allele named cnx5-2 (originated from SALK_039229 line) (supplemental Fig.