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. 2015 Dec;21(12):2103–2118. doi: 10.1261/rna.053934.115

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© 2015 Lin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 4. — Genetic and molecular complementation of yeast t6A mutants with Drosophila orthologs. (A–C) Single complementation tests. The schematic depicts the transformations of cognate yeast genes and their fly orthologs, assayed for complementation of sua5 (A), kae1 (B), and bud32 (C) null mutants. Comparison of single colony sizes indicates substantial growth rescue by fly proteins, although not as robust as the yeast counterparts. (D,E) Double complementation tests. The schematics depict combinations of fly orthologs assayed for complementation of kae1 (D) and bud32 (E) null mutants. Improved growth rescue by supplying combinations of fly factors was noticeable, particularly for Drosophila kae1 + bud32. (F) Primer extension analysis demonstrating molecular rescue of t6A-modified tRNA-Ile by yeast and fly factors. (G) Mass spectrometry analysis of total yeast tRNAs. kae1 mutant yeast has borderline detectable levels of t6A-modified tRNAs, which are restored by complementation with yeast KAE1 and by Drosophila KEOPS genes. Consistent with the behavior in yeast growth (D) and in primer extension assays (F), the combination of Drosophila kae1 + bud32 conferred the best rescue of t6A-modified tRNAs.