Uncoupling the Pleiotropic Phenotypes of clk-1 with tRNA Missense Suppressors in Caenorhabditis elegans

Abstract

clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-toLys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNA Glu genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNA Glu family are expressed in different tissues and at different levels.Suppressor analysis has been routinely used in Caenorhabditis elegans for the study of gene function and the identification of genetic pathway components. Suppressor analysis of nonsense alleles has also led to the discovery of informational suppressors encoding components of the translational machinery, including suppressor tRNAs (for example, references 1, 21, 22, and 40). In fact, C. elegans is the only metazoan from which nonsense tRNA suppressors have been recovered in classic forward genetic screens.We have used a suppressor approach to understand the pleiotropic phenotypes of the C. elegans clk-1 mutants. Mutations in clk-1 are highly pleiotropic, affecting the rates of many physiological processes over a wide range of time scales (41). These mutations result in an average lengthening of the cell cycle of early embryos, of embryonic and postembryonic development, and of the defecation, swimming, and pharyngeal pumping cycles of adults. clk-1 mutations also affect reproductive features, such as the rates of germ line development and egg production (35, 41), and lead to an increased life span (23).clk-1 encodes a highly conserved hydroxylase (9, 18, 29, 36) that is required for the hydroxylation of 5-demethoxyubiquinone to 5-hydroxyubiquinone, which is converted by another enzyme (COQ-3) into ubiquinone (UQ; also called coenzyme Q and CoQ). In the absence of CLK-1, worms are devoid of UQ 9 (the subscript refers to the number of isoprene units in the side chain and is a species-specific trait) (16, 30) and instead accumulate the precursor demethoxyubiquinone (DMQ 9 ) (30). Several observations suggest that DMQ can partially substitute for UQ. ...

Reversal of the Mitochondrial Phenotype and Slow Development of Oxidative Biomarkers of Aging in Long-lived Mclk1+/− Mice

“…Thus, it appears that MCLK1 has an additional function that is unrelated to UQ biosynthesis but responsible for the phenotypes observed in young Mclk1 ϩ/Ϫ mutants. This is consistent with several results from nematodes which also strongly suggest that CLK-1 has other functions (24,25).…”

Early Mitochondrial Dysfunction in Long-lived Mclk1+/- Mice

“…In particular, it was found that both the phenotypically severe allele clk-1(qm30), a partial deletion that does not produce any CLK-1 protein, and the phenotypically weaker allele clk-1(e2519), a Glu to Lys missense mutation that produces wild-type levels of a full-length mutant protein (11), are equally unable to sustain UQ biosynthesis (12). Furthermore, suppression of clk-1(e2519) by a tRNA missense suppressor produces only very small amounts of UQ 9 but results in full phenotypic suppression (13). Together these observations suggest that at least part of the phenotype of clk-1 mutants is not because of the defect in UQ biosynthesis.…”

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

“…However, several lines of evidence argue against CoQ depletion accounting for the effects of statins on mitochondria: (i) supplying CoQ to C. elegans conferred no protection from the toxic effects of statins (CoQ 10 was tested at 50 μg/mL and concentrations of CoQ 9 ranging from 10 to 80 μg/mL were tested, all without effect); (ii) the atfs-1(et15) mutant is not resistant to rotenone, antimycin A, or sodium azide, which are inhibitors of the mitochondrial respiratory chain (25,26) (Fig. S7 B-D); and (iii) it is well known that CoQ is dietarily available to C. elegans that are fed Escherichia coli as in our experiments (27,28) and that very little endogenous CoQ is sufficient for a wild-type phenotype even in the absence of dietary CoQ (29).…”