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The mevalonate pathway regulates microRNA activity in Caenorhabditis elegans - PubMed

  • ️Sun Jan 01 2012

The mevalonate pathway regulates microRNA activity in Caenorhabditis elegans

Zhen Shi et al. Proc Natl Acad Sci U S A. 2012.

Abstract

The mevalonate pathway is highly conserved and mediates the production of isoprenoids, which feed into biosynthetic pathways for sterols, dolichol, ubiquinone, heme, isopentenyl adenine, and prenylated proteins. We found that in Caenorhabditis elegans, the nonsterol biosynthetic outputs of the mevalonate pathway are required for the activity of microRNAs (miRNAs) in silencing their target mRNAs. Inactivation of genes that mediate multiple steps of the mevalonate pathway causes derepression of several miRNA target genes, with no disruption of the miRNA levels, suggesting a role in miRNA-induced silencing complex activity. Dolichol phosphate, synthesized from the mevalonate pathway, functions as a lipid carrier of the oligosaccharide moiety destined for protein N-linked glycosylation. Inhibition of the dolichol pathway of protein N-glycosylation also causes derepression of miRNA target mRNAs. The proteins that mediate miRNA repression are therefore likely to be regulated by N-glycosylation. Conversely, drugs such as statins, which inhibit the mevalonate pathway, may compromise miRNA repression as well as the more commonly considered cholesterol biosynthesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Inactivation of hmgs-1 causes let-7–like phenotypes. (A) Shown are the percentage of animals that burst after the L4-to-adult molt upon treatment with control, alg-1/2, or hmgs-1 RNAi in the indicated genetic background. Inactivation of alg-1/2 or hmgs-1 causes bursting with high penetrance in the wild-type animals but lower penetrance in the lin-42(n1089) or lin-28(n719) loss-of-function mutants. (B) Inactivation of hmgs-1 causes animals to not express col-19::gfp in hyp7 cells at the adult stage. The penetrance of this phenotype is elevated in the let-7(mg279) mutant and decreased in the lin-42(n1089), lin-28(n719), or lin-41(ma104) loss-of-function mutants. Inactivation of alg-1/2 also causes animals to not express col-19::gfp in hyp7 cells; this phenotype is suppressed in lin-42(n1089).

Fig. 2.
Fig. 2.

Inactivation of hmgs-1 causes desilencing of miRNA target genes. (A) Inactivation of hmgs-1 causes defects in the down-regulation of hbl-1::gfp at the L3 stage. This resembles the phenotype caused by mutations in the let-7 family of miRNAs: mir-48, mir-241, and mir-84. The desilencing of hbl-1::gfp upon hmgs-1 RNAi is rescued by supplementation with 2 mM mevalonate. Images were captured using the same exposure settings and processed identically. Arrowheads point to the desilenced hbl-1::gfp in the nuclei of hyp7 cells. (Insets) Nomarski images. (B and C) Immunoblots. Actin was probed as a control for even loading. (B) Inactivation of hmgs-1 causes defects in the down-regulation of lin-14, the target of lin-4 miRNA, at the late L1 stage (lane 5) and early L2 stage (lane 6). Mevalonate supplementation rescues this phenotype (lanes 8 and 9). (C) Inactivation of hmgs-1 does not further desilence lin-14 in the lin-14(n355n679) mutant lacking the lin-14 3′ UTR or the lin-4(e912) null mutant (compare lanes 5 and 6 to lanes 2 and 3). (D) lsy-6 miRNA is expressed in the ASEL but not ASER neuron in the wild type. It is required for ASEL specification, as judged by the expression of lim-6pro::gfp in ASEL, which is promoted by down-regulation of cog-1 by lsy-6. In a sensitized genetic background with a weak allele of lsy-6, ot150, inactivation of alg-1/2 or hmgs-1 significantly enhanced the ASEL specification defect. Supplementing 2 mM mevalonate rescued the phenotype of hmgs-1 inactivation. Brackets indicate statistically significant difference as judged by a two-tailed χ2 test.

Fig. 3.
Fig. 3.

hmgs-1 acts downstream of miRNA biogenesis/accumulation and loading of ALG-1. (A) Shown are the mature miRNA levels in total worm lysate, determined by real-time PCR. The miRNA levels are reduced upon alg-1/2 inactivation but remain unchanged upon hmgs-1 inactivation. (B and C) HA-ALG-1 was immunoprecipitated from animals treated with control or hmgs-1 RNAi, and the level of HA-ALG-1–bound miRNAs was determined. (B) Equal amounts of HA-ALG-1 were purified from control and hmgs-1 RNAi-treated animals. Shown is the Western blot of HA-ALG-1 in total lysate (Upper) and from HA-ALG-1 IP (Lower). (C) Relative levels of let-7, lin-4, and mir-55 bound by HA-ALG-1; they remain unchanged upon hmgs-1 inactivation. In A and C, for each miRNA, the result is shown relative to its level in animals treated with control RNAi. The mean and SD were calculated from three biological replicates. Error bars represent SEM.

Fig. 4.
Fig. 4.

The mevalonate pathway modulates miRNA activity. (A) Inactivation of the mevalonate pathway by either application of fluvastatin, inactivation of hmgs-1 by RNAi, or mutation of hmgr-1, with a low level of mevalonate (1.5 mM) supplied in the medium, causes let-7(mg279) mutants to fail to express col-19::gfp in hyp7 cells. This phenotype can be rescued by supplementing mevalonate [2 mM for hmgs-1 RNAi and 20 mM for the hmgr-1(tm4368) mutant]. (B) Inactivation of the mevalonate pathway by application of fluvastatin or mutation of hmgr-1, with a low amount of mevalonate (1.5 mM) supplied in the medium, causes defects in the down-regulation of hbl-1::gfp (targeted by the let-7 family of miRNAs) at the L3 stage. However, hmgr-1(tm4368) animals growing on high mevalonate (20 mM) show wild-type down-regulation of hbl-1::gfp. Images were captured using the same exposure settings and processed identically. Arrowheads point to the desilenced hbl-1::gfp in the nuclei of hyp7 cells. (Insets) Nomarski images.

Fig. 5.
Fig. 5.

The dolichol pathway for protein N-glycosylation is required for miRNA activity. (A) Inhibiting OST activity by RNAi depletion of any of its five subunits causes defects in the nominal adult-stage up-regulation of col-19::gfp in hyp7 cells in a let-7(mg279) but not wild-type background. (B) Tunicamycin causes defects in the nominal adult-stage up-regulation of col-19::gfp in the let-7(mg279) mutant in a dose-dependent manner. (C) Inhibiting OST activity by RNAi depletion of its catalytic subunit, T12A2.2/STT3, causes defects in the down-regulation of hbl-1::gfp at the L3 stage. Images were captured using the same exposure settings and processed identically. Arrowheads point to the desilenced hbl-1::gfp in the nuclei of hyp7 cells. (Insets) Nomarski images. (D) RNAi depletion of T12A2.2/STT3 enhances the ASEL specification defect in the lsy-6(ot150) mutant but not wild-type genetic background. Brackets indicate a statistically significant difference as judged by a two-tailed χ2 test.

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