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, 15 (3), 207-212

RNA-protein Interaction Detection in Living Cells

Affiliations

RNA-protein Interaction Detection in Living Cells

Muthukumar Ramanathan et al. Nat Methods.

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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
RNA–protein interaction detection (RaPID). (a) Schematic. BoxB RNA stem loops (blue) flank RNA sequence of interest (red). RaPID (λN-HA-BirA*) fusion protein binding to BoxB sites leads to biotinylation of proteins proximal to inserted RNA sequence in living cells grown in biotin-containing media. Streptavidin (SA) beads capture biotinylated protein for MS or western blotting. (b) Quantification of biotinylated RNA pulldowns (bio-EDEN15 and bio-scr control) versus RaPID-Westerns for EDEN15 (n = 3 biologically independent experiments; bars represent mean signal, and error bars denote s.e.m.; one-way ANOVA performed; ****, P < 0.0001). (c) RaPID-MS for EDEN15 identifies CELF1 as binding partner.
Figure 2
Figure 2
Application of RaPID to single-nucleotide human genetic disorders and to viral disease. (a) Schematic of wild-type FTL IRE; circled residues indicate position of previously identified HHCS point mutations; city denotes mutation kindred. (b) RaPID-Western quantified replicates of WT and mutant IREs from HHCS kindreds. IREB2 RaPID signal for each mutant is normalized to IREB2 RaPID signal obtained for wild type and plotted in box graph and serum ferritin levels for each mutant from Allerson et al. are shown in green (n = 3 RaPID-Westerns performed, biologically independent experiments). WT, wild type. (c) GO analysis performed on host proteins found to be bound to ZIKV 5′ and 3′ UTR (SAINT score > 0.9) by RaPID-MS. (d) qPCR measurement of ZIKV viral RNA load in U87 cells (n = 3 biologically independent experiments) comparing control shRNA with QKI shRNA samples. Data normalized to control shRNA. Scatter plots with mean signal and error bars denote s.e.m. (e) Quantification of CVB3 viral RNA load in U87 cells (n = 3 biologically independent experiments) comparing control shRNA with QKI shRNA samples, normalized to control shRNA. Scatter plots with mean signal and error bars denote s.e.m.
Figure 3
Figure 3
Assessment of RaPID specificity by RaPID-MS and identification of RC3H1 binding to the SM1v1 motif. (a) RaPID-MS summary of LC-MS/MS experiments comprising independent two biological replicates for each RNA sequence examined. RNA sequences (rows) and top interacting proteins (columns) are shown with LC-MS/MS SAINT score noted for each protein–RNA motif pair. Syn-EIR represents a synthetic concatamer of the EDEN15, IRE and ROQ CDE motifs. (b) Analysis of TCGA breast cancer patient survival comparing patients with RC3H1 and RC3H2 upregulation (red line) versus without upregulation (blue line). Logrank test P value calculated using cbioportal noted on plot. (c) CLIP-qPCR (n = 3 biologically independent experiments) shows direct interaction between SM1v1 motif and HA-RC3H1 compared to HA-mCherry (P < 0.0001, exact P value = 0.00005891, P values were calculated using unpaired t-test). Scatter plots with mean signal and error bars denote s.e.m.
Figure 4
Figure 4
Faster proximity labeling with engineered biotin ligase BASU. (a) Annotation and comparison of biotin ligases used in the screen. (b) Relative streptavidin signal of total cell lysate with each mutant normalized to streptavidin signal obtained using E. coli BirA*.

Comment in

  • RaPID Hookup
    G Miura. Nat Chem Biol 14 (4), 327. PMID 29556100.

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