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Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics

Nature Geneticsvolume 50pages657661 (2018) | Download Citation


RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as ‘crosstalk with endogenous RBPs’ (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.

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Change history

  • 20 July 2018

    In the version of this article originally published, in the abstract, a single open quotation mark was missing due to a publisher error. The sentence beginning “We present a crosstalk scenario, designated as crosstalk with endogenous RBPs’ (ceRBP), in which…” should have read, “We present a crosstalk scenario, designated as ‘crosstalk with endogenous RBPs’ (ceRBP), in which….” This error has now been corrected in the HTML and PDF versions of the paper.


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We are grateful to C.K. JnBaptiste, S. Garg, and the members of Sharp laboratories for discussions and assistance, and to R.L. Boudreau and B.L. Davidson for siSPOTR analysis. We thank the Robert A. Swanson (1969) Biotechnology Center at the Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology for technical support, specifically G. Paradis, M. Jennings, and M. Saturno-Condon of the Flow Cytometry Core Facility. H.I.S. is supported by the Uehara Memorial Foundation Research Fellowship and the Osamu Hayaishi Memorial Scholarship for Study Abroad. This work was supported by United States Public Health Service Grants R01-GM034277 and R01-CA133404 to P.A.S. from the National Institutes of Health, and by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute.

Author information


  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    • Hiroshi I. Suzuki
    •  & Phillip A. Sharp
  2. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    • Ryan M. Spengler
  3. Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    • Giedre Grigelioniene
    •  & Tatsuya Kobayashi
  4. Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

    • Giedre Grigelioniene
  5. Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden

    • Giedre Grigelioniene
  6. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    • Phillip A. Sharp


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H.I.S. conceived and designed the research, performed computational and experimental analyses, and wrote the manuscript with contributions from all authors. R.M.S. provided data resources. G.G. and T.K. carried out miRNA mutation project. P.A.S. supervised the project and writing of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Phillip A. Sharp.

Integrated supplementary information

  1. Supplementary Figure 1 Effects of overlap between siRNA seeds and known RBP motifs on expression changes of downregulated off-target genes.

    Expression changes of downregulated off-target genes (transcripts with 3´ UTRs containing 7- and 8-mer seed-binding sites and ≤ -0.3 log2 fold change) are shown for two groups. Although not significant, downregulated off-target genes for RBP motif-overlapping siRNAs tended to show relatively larger expression changes relative to those for non-overlapping siRNAs. Statistical significance was assessed using two-sided Wilcoxon signed rank test. n.s.: not significant.

  2. Supplementary Figure 2 Results of DEMETER–ceRBP analysis.

    For several RBPs, distributions of Z-scores of Pearson correlation coefficients for pairs of indicated RBPs and all seeds or motif-overlapping seeds (red: competitive, blue: cooperative) are shown. P values were calculated by one-sided Kolmogorov–Smirnov (K-S) test for either direction depending on the median changes.

  3. Supplementary Figure 3 IGF2BPs target analysis.

    The left plot shows expression changes of IGF2BP1/2/3-bound transcripts defined by PAR-CLIP upon triple knockdown of IGF2BP1/2/3, based on the PAR-CLIP and microarray datasets from Hafner et al. (2010), indicating destabilization of IGF2BP1/2/3-bound transcripts upon their depletion. The middle panel shows expression changes of IGF2BP1/2/3 PAR-CLIP targets upon triple knockout of Igf2bp1/2/3 in mouse mesenchymal stem cells, based on the RNAseq datasets from JnBaptiste et al. (2017). P values were calculated by two-sided Wilcoxon signed rank test with Bonferroni correction. We focused on the representative target HMGA2 and selected shRNAs with IGF2BP2 motif-overlapping seeds and RBP motif non-overlapping seeds for subsequent functional analyses as shown in the right panel.

  4. Supplementary Figure 4 Flow cytometry gating strategies.

    A representative example of gating strategy for MSC.

  5. Supplementary Figure 5 Distribution of DEMETER solutions for shRNAs with RBP motif overlapping seeds.

    Distribution of DEMETER gene solution and seed solutions for shRNAs with seeds overlapping the motifs of indicated RBPs are shown (red: competitive, blue: cooperative). For several competitive RBPs including IGF2BP2 (Fig. 4a, middle), RBM28, ESRP2, and RBFOX2, shRNAs with seeds overlapping their binding motifs seldom have high gene effects and low seed effects, i.e. features of good shRNAs (the top-left region). This trend is not observed for PABPC1 (Fig. 4a, right) and RBM47.

  6. Supplementary Figure 6 Effects of ceRBP crosstalk on DEMETER gene solutions.

    All shRNAs (~100,000) are grouped into three groups according to values of DEMETER seed solutions shown in Fig. 4a (q1: < 0.2, q2: ≥ 0.2 and < 0.6, and q3: ≥ 0.6), and values of DEMETER gene solution are compared. shRNAs with competitive seeds have significantly lower gene effects in a q1 group. P values (versus no overlap) were calculated by two-sided Wilcoxon signed rank test with Bonferroni correction. n.s.: not significant.

  7. Supplementary Figure 7 Effects of ceRBP crosstalk on the performance of shRNAs for cancer essential genes.

    shRNAs for 448 cancer genes differentially required in subsets of 501 cancer cell lines are analyzed, and distribution of DEMETER gene solution and seed solutions for shRNAs without RBP overlapping seeds (left) or with competitive (middle, Z-score > 2) or cooperative (right, Z-score < -2) seeds are shown.

  8. Supplementary Figure 8 Effects of miRNA-type seeds on DEMETER gene solutions.

    All shRNAs are grouped as in Supplementary Fig. 6, and values of DEMETER gene solution are compared. shRNAs with seeds overlapping broadly conserved miRNAs show an approximately twofold decrease in gene effects in a q1 group. P values (versus no overlap) were calculated by two-sided Wilcoxon signed rank test with Bonferroni correction. n.s.: not significant.

  9. Supplementary Figure 9 Overlap between endogenous miRNA seeds and RBP binding motifs.

    A percentage of seeds overlapping the RBP binding motifs is shown. Numbers in parentheses indicate the total number of each small RNA group.

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