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A whole-genome RNAi Screen for C. elegans miRNA pathway genes - PubMed

  • ️Mon Jan 01 2007

A whole-genome RNAi Screen for C. elegans miRNA pathway genes

Devin H Parry et al. Curr Biol. 2007.

Abstract

Background: miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs downregulate target gene expression. An exploration of which protein cofactors are necessary for a miRNA to downregulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes.

Results: A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the downregulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes.

Conclusions: The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or for recognizing the miRNA base-paired to the target mRNA to downregulate translation.

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Figures

Figure 1
Figure 1. let-7 miRNA pathway candidate genes

A. The average burst score from all trials (7–11 trials for each). B. Gene inactivations for which bursting depends on the presence of the let-7(mg279) allele. Positives are defined as a difference of at least 1.5 in average bursting score over 3 trials comparing eri-1(mg366); let-7(mg279) to eri-1(mg366). C. Gene inactivations where bursting is suppressed by lin-41(ma104). Positives which are shaded are defined as a difference of at least 1.5 in average bursting score over 3 trials comparing eri-1(mg366); let-7(mg279) to lin-41(ma104); eri-1(mg366); let-7(mg279). D. Percentage of col-19::GFP;let-7(mg279) worms with disrupted patterns of expression. Standard deviation of three trials is shown in parentheses. Shading indicates a p value ≤ 0.05 determined by Chi square test comparing RNAi feeding to empty vector control. E. Percentage of eri-1(mg366) worms with gaps in adult alae after RNAi feeding. Standard deviation of three trials is shown in parentheses, when appropriate. Shading indicates a p value ≤ 0.05 determined by Chi square test comparing RNAi feeding to empty vector control. F. Shading indicates a perdurance of LIN-14 protein in L2 worms as determined by a comparison on a LIN-14 Western blot to the empty vector-fed control (see Figure 3).

Figure 2
Figure 2. let-7 Northern blot

Total RNA from young adult worms raised for two generations on feeding RNAi probed for let-7. dcr-1 (RNAi) results in an accumulation of precursor and loss of mature let-7 miRNA. Other RNAi treatments do not affect let-7 processing or expression.

Figure 3
Figure 3. LIN-14 Western blots

Protein samples from synchronized L1 and L2 (24 hours at 20 °C) worms are shown for each RNAi treatment. LIN-14 levels drop dramatically from L1 to L2 when fed empty vector control. Due to variations in LIN-14 intensity between blots all comparisons were made to the empty vector control lanes for each blot to determine if LIN-14 down-regulation was affected.

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