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

Nature Methods volume 15, pages 1074–1082 (2018)Cite this article

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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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Fig. 1: Proximity-CLIP scheme.
Fig. 2: Proximity-CLIP accurately identifies proteins and transcripts localized to the nucleus or cytoplasm.
Fig. 3: Analysis of RBP footprints captures short-lived RNA elements and RNA regulatory elements.
Fig. 4: Proximity-CLIP accurately identifies proteins and transcripts localized to cell–cell junctions.

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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.

References

  1. Taliaferro, J. M., Wang, E. T. & Burge, C. B. Genomic analysis of RNA localization. RNA. Biol. 11, 1040–1050 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Lécuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Ephrussi, A., Dickinson, L. K. & Lehmann, R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37–50 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Long, R. M. et al. Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277, 383–387 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Wilk, R., Hu, J., Blotsky, D. & Krause, H. M. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs. Genes Dev. 30, 594–609 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kejiou, N. S. & Palazzo, A. F. mRNA localization as a rheostat to regulate subcellular gene expression. Wiley Interdiscip. Rev. RNA 8, 1–11 (2017).

    Article  CAS  Google Scholar 

  7. Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kaewsapsak, P., Shechner, D. M., Mallard, W., Rinn, J. L. & Ting, A. Y. Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein–RNA crosslinking. eLife 6, e29224 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chen, C.-L. & Perrimon, N. Proximity-dependent labeling methods for proteomic profiling in living cells. Wiley Interdiscip. Rev. Dev. Biol. 6, e272 (2017).

    Article  CAS  Google Scholar 

  10. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Choder, M. mRNA imprinting: additional level in the regulation of gene expression. Cell. Logist. 1, 37–40 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Gerstberger, S., Hafner, M. & Tuschl, T. A census of human RNA-binding proteins. Nat. Rev. Genet. 15, 829–845 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Baltz, A. G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Lee, S. Y. et al. APEX fingerprinting reveals the subcellular localization of proteins of interest. Cell Rep. 15, 1837–1847 (2016).

    Article  CAS  PubMed  Google Scholar 

  16. Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

  17. Slany, A. et al. Contribution of human fibroblasts and endothelial cells to the hallmarks of inflammation as determined by proteome profiling. Mol. Cell. Proteomics 15, 1982–1997 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Corcoran, D. L. et al. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Elkon, R., Ugalde, A. P. & Agami, R. Alternative cleavage and polyadenylation: extent, regulation and function. Nat. Rev. Genet. 14, 496–506 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Tian, B. & Manley, J. L. Alternative cleavage and polyadenylation: the long and short of it. Trends Biochem. Sci. 38, 312–320 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Schueler, M. et al. Differential protein occupancy profiling of the mRNA transcriptome. Genome Biol. 15, R15 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cenik, C. et al. Genome analysis reveals interplay between 5ʹ UTR introns and nuclear mRNA export for secretory and mitochondrial genes. PLoS Genet. 7, e1001366 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Palazzo, A. F. et al. The signal sequence coding region promotes nuclear export of mRNA. PLoS Biol. 5, e322 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Carmody, S. R. & Wente, S. R. mRNA nuclear export at a glance. J. Cell Sci. 122, 1933–1937 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mercer, T. R. et al. Genome-wide discovery of human splicing branchpoints. Genome Res. 25, 290–303 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Valadkhan, S. & Manley, J. L. Splicing-related catalysis by protein-free snRNAs. Nature 413, 701–707 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Luo, W. & Bentley, D. A ribonucleolytic rat torpedoes RNA polymerase II. Cell 119, 911–914 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Vilborg, A., Passarelli, M. C., Yario, T. A., Tycowski, K. T. & Steitz, J. A. Widespread inducible transcription downstream of human genes. Mol. Cell 59, 449–461 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Vilborg, A. et al. Comparative analysis reveals genomic features of stress-induced transcriptional readthrough. Proc. Natl. Acad. Sci. USA 114, E8362–E8371 (2017).

    Article  CAS  Google Scholar 

  30. Almada, A. E., Wu, X., Kriz, A. J., Burge, C. B. & Sharp, P. A. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499, 360–363 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Core, L. J. & Lis, J. T. Transcription regulation through promoter-proximal pausing of RNA polymerase II. Science 319, 1791–1792 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ntini, E. et al. Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality. Nat. Struct. Mol. Biol. 20, 923–928 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Seila, A. C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Affymetrix ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457, 1028–1032 (2009).

  35. Preker, P. et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science 322, 1851–1854 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Cabili, M. N. et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol. 16, 20 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gogakos, T. et al. Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP. Cell Rep. 20, 1463–1475 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Haussecker, D. et al. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16, 673–695 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kumar, P., Anaya, J., Mudunuri, S. B. & Dutta, A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets. BMC Biol. 12, 78 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Xie, X. et al. Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434, 338–345 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bartel, D. P. Metazoan microRNAs. Cell 173, 20–51 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Agarwal, V., Bell, G. W., Nam, J. W. & Bartel, D. P. Predicting effective microRNA target sites in mammalian mRNAs. eLife 4, 1–38 (2015).

    Article  Google Scholar 

  43. Mittelbrunn, M. & Sánchez-Madrid, F. Intercellular communication: diverse structures for exchange of genetic information. Nat. Rev. Mol. Cell Biol. 13, 328–335 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Toyofuku, T. et al. Intercellular calcium signaling via gap junction in connexin-43-transfected cells. J. Biol. Chem. 273, 1519–1528 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Zaidel-Bar, R. Cadherin adhesome at a glance. J. Cell Sci. 126, 373–378 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Guo, Z. et al. E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Sci. Signal. 7, rs7 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Van Itallie, C. M. et al. Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell–cell and cell–substrate adhesion. J. Cell Sci. 127, 885–895 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang, E. T. et al. Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins. Cell 150, 710–724 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Moravcevic, K., Oxley, C. L. & Lemmon, M. A. Conditional peripheral membrane proteins: facing up to limited specificity. Structure 20, 15–27 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. Heller, D., Krestel, R., Ohler, U., Vingron, M. & Marsico, A. ssHMM: extracting intuitive sequence–structure motifs from high-throughput RNA-binding protein data. Nucleic Acids Res. 45, 11004–11018 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Benhalevy, D., Anastasakis, D. & Hafner, M. Proximity-CLIP. Protoc. Exch. https://doi.org/10.1038/protex.2018.115 (2018).

  52. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. van den Ent, F. & Löwe, J. RF cloning: a restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods 67, 67–74 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Spitzer, J., Landthaler, M. & Tuschl, T. Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® recombination cloning technology and Flp-In T-REx® lines. Methods Enzymol. 529, 99–124 (2013).

    Article  CAS  PubMed  Google Scholar 

  56. Gagnon, K. T., Li, L., Janowski, B. A. & Corey, D. R. Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat. Protoc. 9, 2045–2060 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Benhalevy, D., McFarland, H. L., Sarshad, A. A. & Hafner, M. PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites. Methods 118–119, 41–49 (2017).

    Article  CAS  PubMed  Google Scholar 

  58. Hafner, M. et al. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods 58, 164–170 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  60. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

    Article  Google Scholar 

  61. Olarerin-George, A. O. & Jaffrey, S. R. MetaPlotR: a Perl/R pipeline for plotting metagenes of nucleotide modifications and other transcriptomic sites. Bioinformatics 33, 1563–1564 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genomics 15, 284 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).

    Article  CAS  PubMed  Google Scholar 

  65. Huang, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  CAS  Google Scholar 

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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.

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  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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  1. Daniel Benhalevy
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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.

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Correspondence to Markus Hafner.

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Integrated supplementary information

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Supplementary Figure 12

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Table 1

Reporting Summary

Supplementary Protocol

Proximity-CLIP protocol

Supplementary Data 1

Nuclear and cytoplasmic proteomic data and analyses.

Supplementary Data 2

Nuclear and cytoplasmic RNA analyses.

Supplementary Data 3

RNA-seq and CLIP data after raw analysis.

Supplementary Data 4

Cell–cell interface proteomic data and analyses.

Supplementary Data 5

Cell–cell interface RNA analyses.

Supplementary Data 6

UniProt human protein .fasta file configured for MaxQuant analysis.

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Benhalevy, D., Anastasakis, D.G. & Hafner, M. Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments. Nat Methods 15, 1074–1082 (2018). https://doi.org/10.1038/s41592-018-0220-y

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