Capturing the interactome of newly transcribed RNA

Abstract

We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.

Figure 1

Establishment of a new technique to capture the newly transcribed RNA interactome. Schematic representation of the RICK procedure.

Figure 2

Analysis of transcripts isolated by RICK. (a) Distribution of RNA species isolated in a representative RICK experiment. Values for the input sample are also shown. The same RICK data set was used for the analysis in b–d. (b,c) Metagene representation of Pol II (black) occupancy and RICK (green) or oligo(dT) capture (blue) RNA-seq signals at gene promoter or gene body of genes with TR > 4 (n = 5,889) (b) and TR < 4 (n = 6,838) (c). (d) Metagene representation of H3K27ac occupancy (black), H3K4me1 occupancy (magenta), and RICK (green) or oligo(dT) capture (blue) RNA-seq signals at potential enhancer sites; n = 18,272. (e) RT-qPCR analysis of different RNA species isolated by RICK or oligo(dT) capture. ‘RICK control’ indicates samples without EU treatment in RICK experiments; ‘Oligo(dT) control’ indicates samples isolated using beads without oligo(dT) probes in oligo(dT) capture; n = 3 biologically independent experiments and data are shown as the mean ± s.d. P value is shown (Student’s t-test, two tailed).

Figure 3

Analysis of proteins isolated by RICK. (a) Hierarchical clustering of the ion intensities of three different LC-MS/MS experiments (n = 3 biologically independent experiments for both RICK and oligo(dT) capture (with or without EU)), colors in the heatmap indicate the pairwise Pearson correlation between the different data sets (n = 1,169). (b) Venn diagram comparing the 344 RICK-exclusive RBPs and different oligo(dT) capture studies using human cells^,,. The serial RNA interactome capture (serIC) study by Conrad et al. is a human RNA interactome using oligo(dT) capture of the nuclei of K562 cells.

Figure 4

Functional characterization of novel candidate RBPs identified by RICK. (a) GO analysis of the 295 RICK-unique RBPs; the top ten GO terms with the smallest P value in the indicated categories are shown (Fisher’s exact test, one tailed). The number of proteins for each individual term is also shown. Biological processes related to mitosis are marked in red. (b) KEGG pathway analysis of the 295 RICK-unique RBPs. The number of proteins for each individual pathway is also shown; P value is also shown (Fisher’s exact test, one tailed).

Figure 5

Identification of METTL1-interacting RNAs. (a) Venn diagram comparing the 720 high-confidence proteins identified by RICK and the 914 high-confidence proteins identified by polyA-depleted RICK. (b) Venn diagram comparing the 295 RICK-unique RBPs and the 914 high-confidence proteins identified by polyA-depleted RICK. The column indicates distribution of the 710 polyA-depleted RICK proteins compared to high-confidence proteins of different oligo(dT) capture studies (green, proteins present in different oligo(dT) capture data sets^,,,; purple, proteins not present in the same oligo(dT) capture data sets). (c) Distribution of RNA species identified by METTL1 PAR-CLIP sequencing. The average of n = 2 biologically independent experiments is shown. (d) RIP-qPCR analysis of different RNAs binding to METTL1 that were identified in the METTL1 PAR-CLIP sequencing. The enrichments were normalized to input; GFP was used as a negative control. Random hexamers or oligo(dT) primers were used for the reverse transcription as indicated. METTL1-interacting mRNAs are marked in gray as opposed to VTRNA1-3; n = 3 biologically independent experiments, and data are shown as the mean ± s.d.; P value is shown (Student’s t-test, two tailed). (e) The most enriched METTL1-binding motifs on all target RNAs (n = 26,311 and 20,816 for Rep1 and Rep2, respectively) identified in two independent PAR-CLIP sequencing experiments; P value is shown (Fisher’s exact test, one tailed).

Figure 6

Capture of the nascent RNA interactome using RICK. (a) Venn diagram comparing the nascent-enriched RBPs identified with each of the short EU labeling times. (b) GO analysis for the 208 nascent-enriched RBPs; P value in the indicated categories is shown (Fisher’s exact test, one tailed).