daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan

Abstract

The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.

Figure 1. Characteristics of daf-31 mutant dauer larvae.

(A) N2 L3 larva pharynx. (B) N2 dauer larva with fully constricted pharynx. (C) N2 dauer larva with dauer alae along the lateral surface of the cuticle (body beneath focal plane). (D) unc-24(e138)daf-31(m655) dauer larva with fully constricted pharynx. (E) unc-24(e138)daf-31(m655) dauer larva with dauer alae (body beneath focal plane). Arrows indicate isthmus of pharynx in panels A, B and D. Arrowheads indicate dauer alae in panels C and E. N2 and unc-24daf-31/nT1 animals were grown on NG agar plates at 20°C. Unc dauer larvae (unc-24daf-31) were identified after animals were starved. (F–H) Representative pictures showing fat accumulation detected by Sudan Black B in daf-2 mutants (F), N2 (G) and daf-31 mutants (H). N2, daf-2(e1370) and daf-31(m655)IV/nT1[unc-?(n754) let-?](IV;V) synchronized L1 larvae were placed on NG agar plates, incubated at 20°C until they entered L3 or dauer-like stages, then collected for staining. Scale bars: 10 µm.

Figure 2. daf-31 expression pattern in wild-type N2 animals.

GFP expression is under the control of the same 760 bp daf-31 promoter that successfully drove daf-31 gene expression and rescued the dauer-like larval arrest phenotype of daf-31 mutants. (A) daf-31 expression in multiple tissues including pharynx, hypodermis, neurons and intestine. High magnification pictures showing the expression of daf-31 in pharynx (B, indicated by arrowheads), head hypodermal cells (B, indicated by arrows), head neurons (C), hypodermal seam cells (D), tail neurons (E, indicated by arrows) and tail hypodermal cells (E, indicated by arrowheads). Photos in B though E: the upper panels show the GFP signal, the middle panels show the same animals in the same focal planes under Nomarski optics, and the bottom panels show the merged images from the upper and middle panels. For all pictures, the left is anterior and the right is posterior. Scale Bars: 10 µM.

Figure 3. Influence of daf-31 on C. elegans lifespan.

(A) N2 animals treated with daf-31 RNAi have a similar lifespan to those treated with the vector RNAi control. daf-31 RNAi significantly decreases the lifespan of RNAi-sensitive rrf-3 mutants (B) and RNAi-sensitive daf-2 mutants (rrf-3;daf-2) (C). (D) daf-31 overexpression enhances the longevity phenotype of daf-2 mutants. (E) daf-31 RNAi abrogates the effect of daf-31 overexpression on the daf-2 mutant lifespan. (F) daf-16 mutations block the further lifespan extension of daf-2 mutants conferred by daf-31 overexpression.

Figure 4. daf-31 overexpression stimulates the transcriptional activity of DAF-16 without influencing the subcellular localization of DAF-16.

qRT-PCR was performed to measure the mRNA expression level of two DAF-16 target genes, sod-3 and bcmo-2, in indicated strains. Y-axis stands for relative mRNA levels. Daf-31 overexpression up-regulates the expression of sod-3 mRNA in both N2 worms and daf-2 mutants (A), and up-regulates the expression of bcmo-2 mRNA in daf-2 mutants (B) **, P<0.01, ***, P<0.001 (t-test). (C) Representative pictures showing the cytosolic localization of DAF-16 (upper panel) and nuclear localization of DAF-16 (lower panel). Scale Bars: 10 µM. (D) The percentage of worms showing the nuclear localization of DAF-16 in wild-type background (TJ356) is similar to that in daf-31 overexpression animals.