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Conditional gene expression and RNAi using MEC-8–dependent splicing in C. elegans

Nature Methods volume 7, pages 407–411 (2010)Cite this article

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Abstract

We describe a method for conditional regulation of gene expression based on the processing of an intron cassette. The RNA processing factor MEC-8 is necessary for the function of the Caenorhabditis elegans touch receptor neurons; mec-8 mutants are touch insensitive. We show here that this insensitivity involves the loss of MEC-8–dependent splicing of mec-2, which encodes a component of the mechanosensory transduction complex. MEC-8 is needed to remove the ninth intron in mec-2 pre-mRNA to form the longest of three mRNAs, mec-2a. Without MEC-8, splicing causes the termination of the transcript. Inclusion of mec-2 intron 9 is sufficient to convey mec-8–dependent regulation on other genes and, in mec-8(u218ts) mutants, resulted in their temperature-dependent expression. Because mec-8 is expressed ubiquitously in embryos and extensively in larvae, this system should produce temperature-sensitive expression for most genes. As an example, we report a strain that exhibits temperature-dependent RNA interference.

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Figure 1: mec-2a mRNA is not produced in mec-8 mutants.
Figure 2: MEC-8 regulates the alternative splicing of mec-2. (a) mec-2 splicing pattern and MEC-2 proteins.
Figure 3: mec-2 intron 9 conveys MEC-8 dependence.
Figure 4: MEC-8–dependent expression of mec-2intron9∷gfp occurs in many tissues.
Figure 5: mec-2 intron 9–containing strains and strains with missense temperature-sensitive mutations show similar responses to temperature.
Figure 6: Conditional mec-2intron9∷rde-1(+) expression allows for rapid RNAi.

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Acknowledgements

We thank M. Huang for providing valuable insight in the work with mec-2, and L. Chasin, J. Manley and members of the Chalfie laboratory for helpful discussions and comments on the manuscript. This work was supported by US National Institutes of Health grant GM30997 to M.C.

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Author notes

  1. Andrea Calixto & Charles Ma

    Present address: Present addresses: Centro de Envejecimiento y Regeneracion (CARE), Centro de Regulacion Celular y Patologia “Joaquin V. Luco (CRCP), MIFAB, Facultad de Ciencias Biologicas, P. Universidad Catolica de Chile, Alameda 340, Santiago, Chile (A.C.); Phalanx Biotech Group, Palo Alto, California, USA (C.M.).,

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  1. Department of Biological Sciences, Columbia University, New York, New York, USA

    Andrea Calixto, Charles Ma & Martin Chalfie

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  1. Andrea Calixto
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Contributions

A.C., C.M. and M.C. designed experiments; A.C. performed the experiments; A.C. and M.C. wrote the manuscript.

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Correspondence to Martin Chalfie.

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Calixto, A., Ma, C. & Chalfie, M. Conditional gene expression and RNAi using MEC-8–dependent splicing in C. elegans. Nat Methods 7, 407–411 (2010). https://doi.org/10.1038/nmeth.1445

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