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Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence

Nature volume 493, pages 694–698 (2013)Cite this article

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Abstract

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute and Dicer as essential cofactors that process and present small RNAs to their targets. Well-validated small RNA pathway cofactors such as these show distinctive patterns of conservation or divergence in particular animal, plant, fungal and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or short interfering RNA (siRNA)-mediated repression in Caenorhabditis elegans and Drosophila melanogaster1,2, and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction data sets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about one-half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small-RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in those species.

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Figure 1: Phylogenetic profiling analysis shows correlated conservation patterns of C. elegans proteins.
Figure 2: Phylogenetic clusters of candidate small RNA pathway proteins.
Figure 3: Select phylogenetic clusters enriched with hits from proteomic and functional genomic small RNA screens.
Figure 4: Inactivation of genes implicated in RNAi pathways re-animates transgenes that are silenced by RNAi.

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Acknowledgements

We thank T. Duchaine for access to his ERI-1 proteomic data before it was published and to S. Fischer, C. Zhang and T. Montgomery for helpful discussions. The work was supported by NIH GM088565 and the Pew Charitable Trusts (J.K.K.) and NIH GM44619 and GM098647 (G.R.).

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Author notes

  1. Allison C. Billi and Gabriel D. Hayes: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Yuval Tabach, Gabriel D. Hayes, Martin A. Newman, Harrison Gabel, Ravi Kamath, Keren Yacoby, Brad Chapman, Susana M. Garcia, Mark Borowsky & Gary Ruvkun

  2. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Yuval Tabach, Gabriel D. Hayes, Martin A. Newman, Harrison Gabel, Ravi Kamath, Susana M. Garcia, Mark Borowsky & Gary Ruvkun

  3. Life Sciences Institute, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Allison C. Billi & John K. Kim

  4. Department of Human Genetics, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Allison C. Billi & John K. Kim

  5. Broad Institute of MIT and Harvard, Cambridge, 02142, Massachusetts, USA

    Or Zuk

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Contributions

Y.T., J.K.K. and G.R. designed experiments; Y.T. developed analytical tools and analysed data; and Y.T., A.C.B., G.D.H., M.A.N., S.M.G., H.G., R.K. and J.K.K. designed and carried out experiments. O.Z. gave statistical support and conceptual advice. Y.T., K.Y, B.C. and M.B. wrote code. Y.T., A.C.B., J.K.K. and G.R. wrote the paper. G.R. and J.K.K. supervised the project.

Corresponding author

Correspondence to Gary Ruvkun.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7, legends for Supplementary Tables 1-2 and 5-7 (see separate excel files for these Supplementary Tables), Supplementary Tables 3, 4 and 8, Supplementary Methods and Supplementary References. (PDF 4426 kb)

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Tabach, Y., Billi, A., Hayes, G. et al. Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence. Nature 493, 694–698 (2013). https://doi.org/10.1038/nature11779

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