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Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments

Nature Methodsvolume 15pages10741082 (2018) | Download Citation

Abstract

Methods for the systematic study of subcellular RNA localization are limited, and their development has lagged behind that of proteomic tools. We combined APEX2-mediated proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced crosslinking to simultaneously profile the proteome and the transcriptome bound by RNA-binding proteins in any given subcellular compartment. Our approach is fractionation independent and allows study of the localization of RNA processing intermediates, as well as the identification of regulatory RNA cis-acting elements occupied by proteins, in a cellular-compartment-specific manner. We used our method, Proximity-CLIP, to profile RNA and protein in the nucleus, in the cytoplasm, and at cell–cell interfaces. Among other insights, we observed frequent transcriptional readthrough continuing for several kilobases downstream of the canonical cleavage and polyadenylation site and a differential RBP occupancy pattern for mRNAs in the nucleus and cytoplasm. We observed that mRNAs localized to cell–cell interfaces often encoded regulatory proteins and contained protein-occupied CUG sequence elements in their 3′ untranslated region.

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Data availability

NGS sequence data have been deposited in the Gene Expression Omnibus (GEO) under accession GSE110380. Raw blots used to produce Fig. 2b,c and Supplementary Figs. 1a and 3a are provided as Supplementary Fig. 12.

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Acknowledgements

We thank the NHLBI proteomics core and A. Aponte and M. Gucek for mass spectrometry performance and analysis, as well as E. Anderson for additional advice on proteomics data analysis. We thank the NIAMS Genomics Core Facility and G. Gutierrez-Cruz and S. Dell’Orso for sequencing support. We also acknowledge the NIH HPC Biowulf cluster, the NIAMS Biodata Mining and Discovery Section, and S. Brooks, H.-W. Sun, and D. Heller for computational resources and support. pcDNA3 Connexin43-GFP-APEX2 and pCDNA3-APEX2-NES were gifts from A. Ting (Stanford University, Stanford, CA, USA). pFC15A-H2B was a gift from G. Hager (CCR/NCI, Bethesda, MD, USA). Finally, we thank the NIH Medical Arts Branch and A. Hoofring for designing Fig. 1. This work was supported by the Intramural Research Program of the National Institute for Arthritis and Musculoskeletal and Skin Disease.

Author information

Affiliations

  1. Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Disease, Bethesda, MD, USA

    • Daniel Benhalevy
    • , Dimitrios G. Anastasakis
    •  & Markus Hafner

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Contributions

The study was conceived by D.B. and M.H. The methodology was developed by D.B. and D.G.A. Data analysis was carried out by D.B. and D.G.A. D.B. and M.H. wrote the original draft of this manuscript. D.B., D.G.A., and M.H. reviewed and edited the manuscript. M.H. acquired funding for this research, provided resources, and supervised the research.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Markus Hafner.

Integrated supplementary information

  1. Supplementary Figure 1 Pulldown of nuclear and cytoplasmic proteins.

    (a), Streptavidin-HRP stained Western blot from H2B-APEX2 (left panel) and APEX2-NES (right panel) Proximity-CLIP experiments. Streptavidin-bound material was eluted in boiling SDS buffer, supplemented with Biotin, and analyzed by Streptavidin-HRP Western blot. Blots from total cell lysate (T) and flowthrough (FT) served as control for quantitative depletion of biotinylated proteins by affinity chromatography. Note that elution yield for WB analysis is only 10–20% due to resistance of Biotin-Streptavidin interaction to boiling in SDS buffer. Results are representative of two independent experiments with identical results. (b-d), Nuclear and Cytoplasmic proteins Label Free Quantification (LFQ) values were obtained by Mass Spectrometry of Endothelial cells nucleus and cytoplasm fractions (Slany et al., Log manipulated see Ref. #17) and by Proximity-biotinylation (this study). Logarithmic regression lines and R2 are presented on the panels. (b,c), LFQ values of nuclear proteins by Proximity-biotinylation versus LFQ values of nuclear (b), n = 990, r = 0.44, p-value= < 10−5) and cytoplasmic (c), n = 102, r = 0.15, p-value = 0.14) proteins obtained by fractionation. (d,e), LFQ values of cytoplasmic proteins by Proximity-biotinylation versus LFQ values of cytoplasmic (d), n = 104, r = 0.42, p-value = <10−5) and nuclear (e), n = 104, r = 0.08, p-value = 0.25) proteins obtained by fractionation.

  2. Supplementary Figure 2 Analysis of RBP footprints enables the capture of short-lived RNA elements and of RNA regulatory elements.

    (a), Transcription downstream of the Cleavage and Poly-Adenylation site (CPA) is detected in the nucleus, by plotting RBP footprint coverage 2000 bp around the CPA. (b) Genomic DNA contamination in Proximity-CLIP is excluded, revealed by the absence of sequencing reads 2000 bp upstream of the transcriptional start site (TSS). (c), Sense read coverage around the transcription start site (TSS), shows evidence for transcription upstream of TSS, detected only by nuclear Proximity-CLIP: Heatmap of coverage around all TSS (left, note the different color intensity scale for nuclear and cytoplasmic footprints). Sense reads coverage around the genomic coordinates of all TSS, per nuclear and cytoplasmic Proximity-CLIP (top middle and right), and plotted again after removal of the 10% TSS with extreme values (bottom middle and right). (d), Footprints of 20–40 nt length plotted around the pre-miRNAs genomic coordinates ± 500 bp. The peak downstream of pre-miRNA coordinates likely stems from the detection of polycistronic miRNA precursors. (e), Nuclear and cytoplasmic 20–40 nt and 40–70 nt long RNA footprints were separated after ligation of a 3′ adapter by Urea-PAGE gel and visualized by 32p autoradiography. Result represents a single independent experiment. (f-j), Short (20–40 nt) and long (40–70 nt) footprint coverage along tRNAs and tRNA-tails genomic coordinates: (f), Long footprint coverage along intron-less tRNA coordinates ± 30b. (g), Long footprint coverage along coordinates of tRNAs with intron ± 30b. (h), Short footprint coverage along intron-less tRNA coordinates ± 30b. (i), Short footprints coverage along intron-less tRNA tails coordinates ± 100b. (j), Short footprint coverage along coordinates of tRNAs with intron ± 30b.

  3. Supplementary Figure 3 Continuous RBP footprint coverage in the nucleus downstream from the 3′ end of lncRNA NEAT1 to the locus of miRNA-612.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue. Other lncRNAs that nuclear Proximity-CLIP suggested may serve as pri-miRNAs miRs-30a, −1208, −4435 are LINC00472, PVT1 and LINC00152, respectively.

  4. Supplementary Figure 4 KCNQ1OT1 antisense lncRNA transcription initiates at the annotated TSS but decays gradually.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue. LncRNAs NR_039995 and lincMKLN_A1 expression is similarly reduced with distance from the TSS.

  5. Supplementary Figure 5 According to total cell extract RNA-seq, LncRNA XLOC_000304 is expressed at annotated coordinates,, but Proximity-CLIP reveals transcription initiation upstream of the annotated start site as divergent transcription from the CNN3 gene.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue. Grey area zooms on the marked coordinates. We observed a similar expression pattern also for lncRNAs TUG1, ANRIL, NR_015404, CRNDE, AS_GARS and lincMKLN1_A1 divergently transcribed from MORC2, CDKN2A, MAPKAPK5, IRX5, GARS and MKLN1, respectively.

  6. Supplementary Figure 6 XLOC_012192 lncRNA appears as a read-through transcription product of the protein-coding gene CDK12.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue. Similarly, KLRAP1 lncRNA was continuously transcribed downstream of MAGOHB.

  7. Supplementary Figure 7 Nuclear and cytoplasmic RAB30-AS1 exon1 seems differentially or sequentially spliced.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue. Grey area zooms on the marked coordinates.

  8. Supplementary Figure 8 A highly abundant uncharacterized small RNA is detected only as an RBP footprint 7 kb upstream of the TERC lncRNA.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue.

  9. Supplementary Figure 9 Protein occupancy signature suggests XIST is dominantly protein-bound at the 5′ ends of its first and last exons.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue.

  10. Supplementary Figure 10 MALAT1 is highly expressed in HEK293 cells but mascRNA is undetectable.

    IGV tracks including APEX2-NES and H2B-APEX2 Proximity-CLIP reads, coverage and protein-bound clusters, as well as APEX2-NES and H2B-APEX2 Proximity-captured intact RNA and total cell RNA sequencing coverage. Data source, and coverage scales are depicted on the left side. Genomic coordinates and annotated Refseq genes are depicted on the top. Forward reads are colored red, reverse reads are in blue.

  11. Supplementary Figure 11 Cell–cell interface Proximity-CLIP pulldown procedure, functional enrichment analysis of enriched proteins by inclusive analysis, and RRE characterization of occupied 3′ UTR sequence elements in enriched mRNA.

    (a), extracts of 4SU-labeled and crosslinked HEK293 cells expressing APEX2 and supplemented with BP and H2O2 as indicated were subjected to streptavidin affinity chromatography, and bound material was eluted in boiling SDS buffer supplemented with Biotin. Top, Streptavidin-HRP stained Western blot from cell extracts and material eluted from streptavidin beads. Only in the presence of H2O2 and BP protein biotinylation and streptavidin binding was observed. Ponceau S stain of total extract nitrocellulose membranes controlled for protein loading. Bottom, immunoblot for the V5-tag or GFP controlling for tagged APEX2 expression. Streptavidin-HRP blots from total cell extract (T) and flowthrough (FT) control for quantitative depletion of biotinylated proteins by affinity chromatography (bottom right). Note that elution yield for WB analysis is only 10–20%, while elution for MS or RNA analyses in Proximity-CLIP experiments was performed using Trypsin or Proteinase K digestion, respectively. Results are representative of two independent experiments with identical results. (b), functional enrichment analysis of the protein hits list obtained by relaxed parameters analysis of proteins enriched in CNX43-Proximity-CLIP vs. cytoplasmic Proximity-CLIP. Results are representative of two independent experiments with similar results. (c), Weblogo of the RRE enriched in 3′ UTR footprints from the top 400 mRNAs enriched at cell-cell interfaces, generated by ssHMM.

  12. Supplementary Figure 12

    Raw blots and Ponceau-stained membranes used in Fig. 2b,c and Supplementary Figs. 1a and 3a.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Table 1

  2. Reporting Summary

  3. Supplementary Protocol

    Proximity-CLIP protocol

  4. Supplementary Data 1

    Nuclear and cytoplasmic proteomic data and analyses.

  5. Supplementary Data 2

    Nuclear and cytoplasmic RNA analyses.

  6. Supplementary Data 3

    RNA-seq and CLIP data after raw analysis.

  7. Supplementary Data 4

    Cell–cell interface proteomic data and analyses.

  8. Supplementary Data 5

    Cell–cell interface RNA analyses.

  9. Supplementary Data 6

    UniProt human protein .fasta file configured for MaxQuant analysis.

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DOI

https://doi.org/10.1038/s41592-018-0220-y