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RNA–protein interaction detection in living cells

Nature Methods volume 15, pages 207–212 (2018)Cite this article

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A Corrigendum to this article was published on 27 April 2018

This article has been updated

Abstract

RNA–protein interactions play numerous roles in cellular function and disease. Here we describe RNA–protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA–protein interactions in living cells on a timescale as short as 1 min.

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Figure 1: RNA–protein interaction detection (RaPID).
Figure 2: Application of RaPID to single-nucleotide human genetic disorders and to viral disease.
Figure 3: Assessment of RaPID specificity by RaPID-MS and identification of RC3H1 binding to the SM1v1 motif.
Figure 4: Faster proximity labeling with engineered biotin ligase BASU.

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Change history

  • 20 April 2018

    In the version of this Article originally published, an amino acid substitution introduced into the B. subtilis biotin ligase sequence was incorrectly written as R142G. The correct mutation is R124G. This error has been corrected in the HTML and PDF versions of the paper.

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Acknowledgements

We thank C. Adams, R. Leib and Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry for help with mass spectrometry. We also thank P. Oikonomou and S. Tavazoie for providing position weight matrixes. We thank A. Fire, H. Chang, J. Elias, P. Sarnow, R. Flynn, J. Quinn, A. Bhaduri, A. Rubin and M. Kay for presubmission review. We thank L. Morcom and P. Bernstein for expert administrative assistance and members of the Khavari lab for helpful discussions. This work was supported by the US VA Office of Research and Development, by NIH AR49737, and NIH AR43799 (P.A.K.). The project described was supported by Award Number S10RR027425 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

Author information

Authors and Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Muthukumar Ramanathan, Deepti S Rao, Poornima H Neela, Brian J Zarnegar, Smarajit Mondal, Joanna R Kovalski, Zurab Siprashvili & Paul A Khavari

  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Karim Majzoub & Jan E Carette

  3. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA

    Julien G Roth, Hui Gai & Theo D Palmer

  4. Veterans Affairs Palo Alto Healthcare System, Palo Alto, California, USA

    Paul A Khavari

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  1. Muthukumar Ramanathan
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Contributions

M.R. designed and executed experiments, analyzed the data and wrote the manuscript. K.M. designed and performed viral replication assays. J.G.R., H.G., D.S.R., J.R.K., P.H.N., B.J.Z., Z.S. and S.M. performed experiments. J.E.C. and T.D.P. supervised work. P.A.K. designed experiments, analyzed data, and wrote the manuscript.

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Correspondence to Paul A Khavari.

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Integrated supplementary information

Supplementary Figure 1 Validation of RaPID with EDEN15

(a) Full Western Blot of conventional biotinylated RNA pulldown with EDEN15 and scrambled control (n=3 biologically independent experiments, representative image shown) (b) Schema of motif plasmid. Motif along with BoxB sites added to 3′UTR of eGFP (c) Quantification of motif plasmid expression in HEK293T cells by qPCR with primers against eGFP. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (d) RaPID-Western of EDEN15 vs two scrambled sequence controls of identical nucleotide composition (scr1 and scr2). HA recognizes the epitope tagged LN-HA-BirA* fusion, which serves as a self-biotinylated streptavidin pulldown internal control (n=3 biologically independent experiments, representative image shown)

Source data

Supplementary Figure 2 RaPID-Western IRE-IREB2 binding with HHCS mutants

(a) RaPID-Western blots of WT IRE and HHCS FTL IRE mutants. Western blotting of IREB2 pulldown, RaPID (λN-HA-BirA*) fusion protein, which serves as an self-biotinylated internal control detected by HA antibody, and IREB2 in the lysate are shown. (n=3 biologically independent experiments, representative image shown) (b) Quantification of motif plasmid expression in HEK293T cells by qPCR with primers against eGFP. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m)

Source data

Supplementary Figure 3 RaPID detects disruption in IRE-IRP1 interaction upon drug treatment

RaPID-Western of IRE motif with treatment (deferoxamine [DFO] 100uM, dimethyl sulfoxide [DMSO] 0.1%, ferric ammonium citrate [FAC] 100uM). (a) RaPID-Western with IRE blotting for IRP1 with different drug treatments. (n=3 biologically independent experiments, representative image shown) (b) Quantification of RaPID-Westerns with drug treatments. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (c) Full Western blot of representative image shown

Source data

Supplementary Figure 4 Analysis of RaPID-MS ZIKV UTR bound proteins

(a) GO Analysis performed on downregulated genes in ZIKV infected NPCs. RNA-Seq data used for analysis obtained from Tang et al.24 (b) Tissue enrichment score plot (please see methods) of RaPID-MS ZIKV UTR bound proteins. (c) Western Blot of U87 cells comparing efficacy of knockdown of QKI using scrambled with QKI shRNA 1 and QKI shRNA 2. (n=3 biologically independent experiments, representative image shown) (d) Quantification of QKI knockdown in U87 cells with Ctrl shRNA, QKI shRNA 1 and QKI shrNA 2. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (e) Full Western blot of representative image shown

Source data

Supplementary Figure 5 QKI Protein levels decrease as NPCs differentiate to Neurons

(a) hiPSC colonies exhibit the pluripotency marker Oct4 (Green). (b) Day 21 NPCs are organized in a radial arrangement around the adherens junction marker NCad (Red) and are positive for the neuroectodermal marker Pax6 (Green) (c) Day 45 neurons are positive for the neuronal markers Tuj1 (Red) and NeuN (Green) (d) Western Blot of QKI comparing levels in hiPSC derived NPCs and neurons. (n=3 biologically independent experiments, representative image shown) (e) Quantification of QKI levels in hiPSC derived NPCs and neurons. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (f) Full Western blot of representative image shown

Source data

Supplementary Figure 6 RaPID-MS SAINT score against FC plot for 7 RNA motifs in Fig 3A

Supplementary Figure 7 RC3H1&2 upergulation is correlated with poor survival outcome in breast cancer

RC3H1&2 upregulation correlated with poor survival outcomes in breast cancer (a) Breast cancer patient survival as a function of RC3H1 upregulation (red line) versus control (blue line). TCGA data - Breast Invasive Carcinoma (TCGA, Provisional) accessed on 5/2/2016 via cBioPortal with query settings of copy number alterations and mRNA expression (z-score>3). Logrank Test P-value calculated by cbioportal noted on plot (b) Breast cancer patient survival as a function of RC3H2 upregulation (red line) versus control (blue line). TCGA data - Breast Invasive Carcinoma (TCGA, Provisional) accessed on 5/2/2016 via cBioPortal with query settings of copy number alterations and mRNA expression (z-score>3).Logrank Test P-value calculated by cbioportal noted on plot (c) Breast cancer patient survival as a function of RC3H1 and RC3H2 upregulation (red line) versus control (blue line). TCGA data - Breast Invasive Carcinoma (TCGA, Provisional) accessed on 5/2/2016 via cBioPortal with query settings of copy number alterations and mRNA expression (z-score>3). Upregulation of RC3H1 and RC3H2 is mutually exclusive (Log Odds Ratio <-3 & Fisher Exact test p-value 0.035). Logrank Test P-value calculated by cbioportal noted on plot

Supplementary Figure 8 Structure and Sequences of Biotin ligases

Structure and sequences of biotin ligases (a) E. coli BirA (PDB:1BIB) annotated with color-coded motifs (RBAM, ABM, AM & PTAM) using MacPyMol (b) Sequences of RBAM, ABM, AM & PTAM in E. coli BirA (c) Sequence alignment of screened biotin ligases (A. aeolicus, Rhodanobacter spp., Tanerella spp., T. brucei) with motifs (RBAM, ABM, AM & PTAM) (d) Sequence alignment of N terminal truncated B. subtilis biotin ligase (67-325) with motifs (RBAM, ABM, AM & PTAM) (e) Alignment of B. subtilis biotin ligase and E. coli BirA*. Mutations made at C-terminus of B. subtilis (67-325) highlighted in red boxes to create BASU with SLR terminal end similar to E. coli BirA*.

Supplementary Figure 9 Expression and Streptavidin signal of HA-tagged mutant BirA*s

(A) HA signal of total cell lysate with each mutant biotin ligase (lower boxed area) transfected in HEK293T cells. Vinculin is blotted (upper boxed area) and used as loading control. (n=3 biologically independent experiments, representative image shown) (B) Streptavidin signal of total cell lysate with each mutant biotin ligase (n=3 biologically independent experiments, representative image shown)

Supplementary Figure 10 Evaluating application of BASU in RaPID with varying labeling timss

(a) RaPID-Western blots of TNF-CDE at different labeling time points ranging from 0min (unlabeled) to 1hour labeling. Western blotting of RC3H1 pulldown, RaPID (λN-HA-BirA*) fusion protein, which serves as an self-biotinylated internal control detected by HA antibody, and RC3H1 in the lysate are shown. (n=3 biologically independent experiments, representative image shown) (b) Full Western blot of representative image shown

Supplementary Figure 11 Higher signal-to-noise ratio with BASU shorter labeling

(a) Quantification of motif plasmid expression in HEK293T cells by qPCR with primers against eGFP. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (b) RaPID-Western of TNF-CDE vs two scrambled sequence controls (scr1 and scr2). HA recognizes the epitope tagged LN-HA-BioID2* fusion, which serves as a self-biotinylated streptavidin pulldown internal control. Labeling performed for 1 min (n=3 biologically independent experiments, representative image shown) (c) RaPID-Western of TNF-CDE vs two scrambled sequence controls (scr1 and scr2). HA recognizes the epitope tagged LN-HA-BASU* fusion, which serves as a self-biotinylated streptavidin pulldown internal control. Labeling performed for 1 min (n=3 biologically independent experiments, representative image shown) (d) Quantification of RaPID-Westerns with BASU 1 min labeling. (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (e) RaPID-Western blots of TNF-CDE and scrambled controls using conventional E. coli BirA* with 18hrs of labeling. Western blotting of RC3H1 pulldown, RaPID (λN-HA-BirA*) fusion protein, which serves as an self-biotinylated internal control detected by HA antibody, and RC3H1 in the lysate are shown. (n=3 biologically independent experiments, representative image shown) (f) Quantification of RaPID-Western of TNF-CDE and scrambled controls using conventional E. coli BirA* with 18hrs of labeling (n=3 biologically independent experiments, bars represent mean signal and error bars denote s.e.m) (g) Signal-to-noise ratio (SNR) comparison between E. coli BirA* at 18hrs and BASU at 1min using TNF-CDE vs scrambled control RNA motifs (h) RaPID-MS SAINT score vs Fold Change (FC) plot for EDEN15 with BASU at labeling time of 30 minutes (i) RaPID-MS SAINT score vs Fold Change (FC) plot for IRE (UTRP 35) with BASU at labeling time of 30 minutes.

Source data

Supplementary Figure 12 Characterization and comparison of E.coli BirA* and BASU

(a) Immunoflourescence of streptavidin in 3D organotypic human skin tissue expressing BASU or E.coli BirA* taken at timepoint 0 min (no biotin labeling) and 30mins (30mins of biotin labeling). (b) Western Blot of streptavidin in 3D organotypic human skin tissue expressing BASU or E.coli BirA* taken at timepoint 0 min (no biotin labeling) and 30mins (30mins of biotin labeling). (n=3 biologically independent experiments, representative image shown) (c) Comparison of gain in streptavidin signal between E.coli BirA* and BASU. Normalized Streptavidin levels (Streptavidin level in Western Blot/ HA levels in Western Blot) were obtained for E.coli BirA* and BASU at each timepoint. The gain in streptavidin signal was obtained by subtracting Normalized Streptavidin levels at timepoint 0 min from timepoint 30mins for E.coli BirA* and BASU.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Notes 1–2 (PDF 3022 kb)

Life Sciences Reporting Summary (PDF 132 kb)

Supplementary Protocol

RaPID-Western (PDF 214 kb)

Supplementary Table 2

EDEN15 RaPID-MS in HEK293T cells. (XLSX 216 kb)

Supplementary Table 3

EDEN15 RaPID-MS in Huh7 cells. (XLSX 102 kb)

Supplementary Table 4

FTL IRE RaPID-MS in HEK293T cells. (XLSX 298 kb)

Supplementary Table 5

ZIKV 5′UTR RaPID-MS in HEK293T cells. (XLSX 271 kb)

Supplementary Table 6

ZIKV 3′UTR RaPID-MS in HEK293T cells. (XLSX 255 kb)

Supplementary Table 7

ZIKV UTR host proteins DAVID GO Term Analysis. (XLSX 83 kb)

Supplementary Table 8

Tissue Protein Level data for ZIKV UTR interacting proteins. (XLSX 47 kb)

Supplementary Table 9

Histone Stem Loop (UTRP11) RaPID-MS in HEK293T cells. (XLSX 214 kb)

Supplementary Table 10

p38_NRE RaPID-MS in HEK293T cells. (XLSX 179 kb)

Supplementary Table 11

PPP1R3C (UTRP30) RaPID-MS in HEK293T cells. (XLSX 220 kb)

Supplementary Table 12

Synthetic-EIR concatamer RaPID-MS in HEK293T cells. (XLSX 204 kb)

Supplementary Table 13

SM1v1 RaPID-MS in HEK293T cells. (XLSX 218 kb)

Supplementary Table 14

BASU EDEN15 RaPID-MS in HEK293T cells at 30 minutes. (XLSX 184 kb)

Supplementary Table 15

FTL IRE RaPID-MS in HEK293T cells at 30 minutes. (XLSX 208 kb)

Supplementary Table 16

List of Background proteins in HEK293T cells (XLSX 74 kb)

Supplementary Table 17

RNA motif sequences utilized in RaPID. (XLSX 58 kb)

Supplementary Table 18

Composition of buffers used in RaPID. (XLSX 41 kb)

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Ramanathan, M., Majzoub, K., Rao, D. et al. RNA–protein interaction detection in living cells. Nat Methods 15, 207–212 (2018). https://doi.org/10.1038/nmeth.4601

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