ABSTRACT: The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. numerous molecules serving as valuable therapeutics. The marine natural product girolline has been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A. Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in ribosome-associated quality control (RQC) and is important in maintaining the efficiency of translational progress. Girolline, therefore, provides a potent tool compound for understanding the interplay between protein production and quality control in a physiological setting and offers a new and selective means of modulating gene expression. numerous molecules serving as valuable therapeutics. The marine natural product girolline has27 been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general28 translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A.29 Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on30 AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in31 ribosome-associated quality control (RQC) and is important in maintaining the efficiency of32 translational progress. Girolline, therefore, provides a potent tool compound for understanding33 the interplay between protein production and quality control in a physiological setting and offers34 a new and selective means of modulating gene expression.
