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Discovery of Plasma Membrane-Associated RNAs through APEX-seq

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Abstract

In addition to nucleic acids, a variety of other biomolecules have also been found on the plasma membrane. Although researchers have realized that RNA has the ability to bind to membrane vesicles in vitro, little is known about whether and how RNA connects to the plasma membrane of the cell. The combination of high-throughput sequencing and in situ labeling methods provides an innovative approach for large-scale identification of subcellular RNAs. Here, we applied the recently published method APEX-seq and identified 75 RNAs related to the plasma membrane, in which lncRNA PMAR72 (plasma membrane-associated RNA AL121772.1) has a considerable affinity with sphingomyelin (SM) and localizes within distinct membrane foci. Our findings will provide some new evidence to elaborate the relationship between RNA and the plasma membrane of mammalian cells.

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Data Availability

APEX-seq data have been deposited in the NCBI GEO under accession number GSE166725.

References

  1. Dipalo, M., Caprettini, V., Bruno, G., Caliendo, F., Garma, L. D., Melle, G., Dukhinova, M., Siciliano, V., Santoro, F., & De Angelis, F. (2019). Membrane poration mechanisms at the cell-nanostructure interface. Advanced Biosystems, 3, e1900148.

    Article  PubMed  Google Scholar 

  2. Avota, E., de Lira, M. N., & Schneider-Schaulies, S. (2019). Sphingomyelin breakdown in T cells: role of membrane compartmentalization in T cell signaling and interference by a pathogen. Frontiers in Cell and Developmental Biology, 7, 152.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Honigmann, A., & Pralle, A. (2016). Compartmentalization of the cell membrane. Journal of Molecular Biology, 428, 4739–4748.

    Article  CAS  PubMed  Google Scholar 

  4. Doktorova, M., Symons, J. L., & Levental, I. (2020). Structural and functional consequences of reversible lipid asymmetry in living membranes. Nature Chemical Biology, 16, 1321–1330.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pace, H., Simonsson Nystrom, L., Gunnarsson, A., Eck, E., Monson, C., Geschwindner, S., Snijder, A., & Hook, F. (2015). Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes. Analytical Chemistry, 87, 9194–9203.

    Article  CAS  PubMed  Google Scholar 

  6. Xu, G., Goonatilleke, E., Wongkham, S., & Lebrilla, C. B. (2020). Deep structural analysis and quantitation of O-linked glycans on cell membrane reveal high abundances and distinct glycomic profiles associated with cell type and stages of differentiation. Analytical Chemistry, 92, 3758–3768.

    Article  CAS  PubMed  Google Scholar 

  7. Abe, M., & Kobayashi, T. (2017). Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis. Methods in Cell Biology, 137, 15–24.

    Article  CAS  PubMed  Google Scholar 

  8. Leung, S. S. W., Brewer, J., Bagatolli, L. A., & Thewalt, J. L. (2019). Measuring molecular order for lipid membrane phase studies: linear relationship between Laurdan generalized polarization and deuterium NMR order parameter. Biochimica et Biophysica Acta - Biomembranes, 1861, 183053.

    Article  CAS  PubMed  Google Scholar 

  9. Beliaev, N. D., Budker, V. G., Gorokhova, O. E., & Sokolov, A. V. (1988). [Mg2+-dependent interaction of DNA with eukaryotic cells]. Molecular Biology, 22, 1667–1672.

    CAS  Google Scholar 

  10. Chakrabarti, A. C., Breaker, R. R., Joyce, G. F., & Deamer, D. W. (1994). Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. Journal of Molecular Evolution, 39, 555–559.

    Article  CAS  PubMed  Google Scholar 

  11. Gruzdev, A. D., Khramtsov, V. V., Weiner, L. M., & Budker, V. G. (1982). Fluorescence polarization study of the interaction of biopolymers with liposomes. FEBS Letters, 137, 227–230.

    Article  CAS  PubMed  Google Scholar 

  12. Khvorova, A., Kwak, Y. G., Tamkun, M., Majerfeld, I., & Yarus, M. (1999). RNAs that bind and change the permeability of phospholipid membranes. Proceedings of the National Academy of Sciences of the United States of America, 96, 10649–10654.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vlassov, A., Khvorova, A., & Yarus, M. (2001). Binding and disruption of phospholipid bilayers by supramolecular RNA complexes. Proceedings of the National Academy of Sciences of the United States of America, 98, 7706–7711.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Janas, T., & Yarus, M. (2003). Visualization of membrane RNAs. RNA, 9, 1353–1361.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Janas, T., Janas, T., & Yarus, M. (2006). Specific RNA binding to ordered phospholipid bilayers. Nucleic Acids Research, 34, 2128–2136.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Block, K. F., Puerta-Fernandez, E., Wallace, J. G., & Breaker, R. R. (2011). Association of OLE RNA with bacterial membranes via an RNA-protein interaction. Molecular Microbiology, 79, 21–34.

    Article  CAS  PubMed  Google Scholar 

  17. Lin, A., Li, C., Xing, Z., Hu, Q., Liang, K., Han, L., Wang, C., Hawke, D. H., Wang, S., Zhang, Y., Wei, Y., Ma, G., Park, P. K., Zhou, J., Zhou, Y., Hu, Z., Zhou, Y., Marks, J. R., Liang, H., Hung, M. C., Lin, C., & Yang, L. (2016). The LINK-A lncRNA activates normoxic HIF1alpha signalling in triple-negative breast cancer. Nature Cell Biology, 18, 213–224.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lin, A., Hu, Q., Li, C., Xing, Z., Ma, G., Wang, C., Li, J., Ye, Y., Yao, J., Liang, K., Wang, S., Park, P. K., Marks, J. R., Zhou, Y., Zhou, J., Hung, M. C., Liang, H., Hu, Z., Shen, H., Hawke, D. H., Han, L., Zhou, Y., Lin, C., & Yang, L. (2017). The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors. Nature Cell Biology, 19, 238–251.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang, N., Fan, X., Zaleta-Rivera, K., Nguyen, T. C., Zhou, J., Luo, Y., Gao, J., Fang, R. H., Yan, Z., Chen, Z. B., Zhang, L., & Zhong, S. (2020). Natural display of nuclear-encoded RNA on the cell surface and its impact on cell interaction. Genome Biology, 21, 225.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hung, V., Zou, P., Rhee, H. W., Udeshi, N. D., Cracan, V., Svinkina, T., Carr, S. A., Mootha, V. K., & Ting, A. Y. (2014). Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Molecular Cell, 55, 332–341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fazal, F. M., Han, S., Parker, K. R., Kaewsapsak, P., Xu, J., Boettiger, A. N., Chang, H. Y., & Ting, A. Y. (2019). Atlas of subcellular RNA localization revealed by APEX-Seq. Cell, 178, 473–490 e426.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, D., Paggi, J. M., Park, C., Bennett, C., & Salzberg, S. L. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nature Biotechnology, 37, 907–915.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liao, Y., Smyth, G. K., & Shi, W. (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Research, 47, e47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. McCarthy, D. J., Chen, Y., & Smyth, G. K. (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research, 40, 4288–4297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Frankish, A., Diekhans, M., Ferreira, A. M., Johnson, R., Jungreis, I., Loveland, J., Mudge, J. M., Sisu, C., Wright, J., Armstrong, J., Barnes, I., Berry, A., Bignell, A., Carbonell Sala, S., Chrast, J., Cunningham, F., Di Domenico, T., Donaldson, S., Fiddes, I. T., Garcia Giron, C., Gonzalez, J. M., Grego, T., Hardy, M., Hourlier, T., Hunt, T., Izuogu, O. G., Lagarde, J., Martin, F. J., Martinez, L., Mohanan, S., Muir, P., Navarro, F. C. P., Parker, A., Pei, B., Pozo, F., Ruffier, M., Schmitt, B. M., Stapleton, E., Suner, M. M., Sycheva, I., Uszczynska-Ratajczak, B., Xu, J., Yates, A., Zerbino, D., Zhang, Y., Aken, B., Choudhary, J. S., Gerstein, M., Guigo, R., Hubbard, T. J. P., Kellis, M., Paten, B., Reymond, A., Tress, M. L., & Flicek, P. (2019). GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res, 47, D766–D773.

    Article  CAS  PubMed  Google Scholar 

  27. Yu, Z., Huang, Z., & Lung, M. L. (2013). Subcellular fractionation of cultured human cell lines. Bio-protocol, 3, e754.

    Article  Google Scholar 

  28. Lemaire-Vieille, C., Gagnon, J., & Cesbron, J.-Y. (2013). Protein flotation assay to isolate lipids rafts from soft tissue or cells. Bio-protocol, 3, e854.

    Article  Google Scholar 

  29. Hung, V., Lam, S. S., Udeshi, N. D., Svinkina, T., Guzman, G., Mootha, V. K., Carr, S. A., and Ting, A. Y. (2017) Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. Elife 6, e24463

  30. Benhalevy, D., Anastasakis, D. G., & Hafner, M. (2018). Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments. Nature Methods, 15, 1074–1082.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

  32. Zlatkine, P., Mehul, B., & Magee, A. I. (1997). Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. Journal of Cell Science, 110(Pt 5), 673–679.

    Article  CAS  PubMed  Google Scholar 

  33. Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J., & Meyer, T. (2005). An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nature Methods, 2, 415–418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Taube, R., Zhu, Q., Xu, C., Diaz-Griffero, F., Sui, J., Kamau, E., Dwyer, M., Aird, D., & Marasco, W. A. (2008). Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles. PLoS One, 3, e3181.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Jing, M., Zhang, P., Wang, G., Feng, J., Mesik, L., Zeng, J., Jiang, H., Wang, S., Looby, J. C., Guagliardo, N. A., Langma, L. W., Lu, J., Zuo, Y., Talmage, D. A., Role, L. W., Barrett, P. Q., Zhang, L. I., Luo, M., Song, Y., Zhu, J. J., & Li, Y. (2018). A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nature Biotechnology, 36, 726–737.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Watson, H. (2015). Biological membranes. Essays in Biochemistry, 59, 43–69.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Wang, P., Tang, W., Li, Z., Zou, Z., Zhou, Y., Li, R., Xiong, T., Wang, J., & Zou, P. (2019). Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nature Chemical Biology, 15, 1110–1119.

    Article  CAS  PubMed  Google Scholar 

  38. Pauling, L., & Corey, R. B. (1953). A proposed structure for the nucleic acids. Proceedings of the National Academy of Sciences of the United States of America, 39, 84–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Allemand, J. F., Bensimon, D., Lavery, R., & Croquette, V. (1998). Stretched and overwound DNA forms a Pauling-like structure with exposed bases. Proceedings of the National Academy of Sciences of the United States of America, 95, 14152–14157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, D. J., & Day, L. A. (1994). Pf1 virus structure: helical coat protein and DNA with paraxial phosphates. Science, 265, 671–674.

    Article  CAS  PubMed  Google Scholar 

  41. Budker, V. G., Kazatchkov, Y. A., & Naumova, L. P. (1978). Polynucleotides adsorb on mitochondrial and model lipid membranes in the presence of bivalent cations. FEBS Letters, 95, 143–146.

    Article  CAS  PubMed  Google Scholar 

  42. Strom, A. R., Emelyanov, A. V., Mir, M., Fyodorov, D. V., Darzacq, X., & Karpen, G. H. (2017). Phase separation drives heterochromatin domain formation. Nature, 547, 241–245.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhou, H., Song, Z., Zhong, S., Zuo, L., Qi, Z., Qu, L. J., & Lai, L. (2019). Mechanism of DNA-induced phase separation for transcriptional repressor VRN1. Angewandte Chemie, 58, 4858–4862.

    Article  CAS  PubMed  Google Scholar 

  44. Suleymanoglu, E. (2017). Mg(2+)-induced DNA compaction, condensation, and phase separation in gene delivery vehicles based on zwitterionic phospholipids: a dynamic light scattering and surface-enhanced Raman spectroscopic study. Journal of Biological Inorganic Chemistry, 22, 1165–1177.

    Article  CAS  PubMed  Google Scholar 

  45. Chong, P. A., Vernon, R. M., & Forman-Kay, J. D. (2018). RGG/RG motif regions in RNA binding and phase separation. Journal of Molecular Biology, 430, 4650–4665.

    Article  CAS  PubMed  Google Scholar 

  46. Ries, R. J., Zaccara, S., Klein, P., Olarerin-George, A., Namkoong, S., Pickering, B. F., Patil, D. P., Kwak, H., Lee, J. H., & Jaffrey, S. R. (2019). m(6)A enhances the phase separation potential of mRNA. Nature, 571, 424–428.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fox, A. H., Nakagawa, S., Hirose, T., & Bond, C. S. (2018). Paraspeckles: where long noncoding RNA meets phase separation. Trends in Biochemical Sciences, 43, 124–135.

    Article  CAS  PubMed  Google Scholar 

  48. Jain, A., & Vale, R. D. (2017). RNA phase transitions in repeat expansion disorders. Nature, 546, 243–247.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yamazaki, T., Souquere, S., Chujo, T., Kobelke, S., Chong, Y. S., Fox, A. H., Bond, C. S., Nakagawa, S., Pierron, G., & Hirose, T. (2018). Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation. Molecular Cell, 70, 1038–1053 e1037.

    Article  CAS  PubMed  Google Scholar 

  50. Gilbert, W. (1986). Origin of life: the RNA world. Nature, 319, 618–618.

    Article  Google Scholar 

  51. Joyce, G. F. (1989). RNA evolution and the origins of life. Nature, 338, 217–224.

    Article  CAS  PubMed  Google Scholar 

  52. Yin, H., Xiong, G., Guo, S., Xu, C., Xu, R., Guo, P., & Shu, D. (2019). Delivery of anti-miRNA for triple-negative breast cancer therapy using RNA nanoparticles targeting stem cell marker CD133. Molecular Therapy, 27, 1252–1261.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Pi, F., Binzel, D. W., Lee, T. J., Li, Z., Sun, M., Rychahou, P., Li, H., Haque, F., Wang, S., Croce, C. M., Guo, B., Evers, B. M., & Guo, P. (2018). Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nature Nanotechnology, 13, 82–89.

    Article  CAS  PubMed  Google Scholar 

  54. Li, H., Wang, S., Ji, Z., Xu, C., Shlyakhtenko, L. S., & Guo, P. (2019). Construction of RNA nanotubes. Nano Research, 12, 1952–1958.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Liang, C., Weitao, T., Zhou, L., & Guo, P. (2020). Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine. Science China Life Sciences, 63, 1103–1129.

    Article  CAS  PubMed  Google Scholar 

  56. Guo, S., Xu, C., Yin, H., Hill, J., Pi, F., & Guo, P. (2020). Tuning the size, shape and structure of RNA nanoparticles for favorable cancer targeting and immunostimulation. Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology, 12, e1582.

    Article  CAS  PubMed  Google Scholar 

  57. Wang, H., Ellipilli, S., Lee, W. J., Li, X., Vieweger, M., Ho, Y. S., & Guo, P. (2020). Multivalent rubber-like RNA nanoparticles for targeted co-delivery of paclitaxel and MiRNA to silence the drug efflux transporter and liver cancer drug resistance. Journal of Controlled Release, 330, 173–184.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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Acknowledgements

This research was supported by grants from the National Key Research and Development Program of the Ministry of Science and Technology of China (2018YFA0106901, 2017YFA0505400, 2019YFA0904201), as well as grants from the National Natural Science Foundation of China (81871898, 31971200, 21927814). We are very grateful to Prof. Pingsheng Liu and Dr. Dongpeng Wang from Institute of Biophysics, Chinese Academy of Sciences for discussions and comments. We thank staffs from Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences for technical assistance. Data analysis and computing resource was supported by Center for Big Data Research in Health (http://bigdata.ibp.ac.cn), Institute of Biophysics, Chinese Academy of Sciences.

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  1. These authors contributed equally: Erzhong Wu, Xuzhen Guo, Xueyi Teng

Authors and Affiliations

  1. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China

    Erzhong Wu, Xuzhen Guo, Xueyi Teng, Ruijin Zhang, Fahui Li, Ya Cui, Dongdong Zhang, Qinghua Liu, Jianjun Luo, Jiangyun Wang & Runsheng Chen

  2. University of Chinese Academy of Sciences, 100049, Beijing, China

    Erzhong Wu, Xuzhen Guo, Xueyi Teng, Ruijin Zhang, Jianjun Luo, Jiangyun Wang & Runsheng Chen

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  1. Erzhong Wu
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Contributions

R.C. initiated the project, J.L., J.W., E.W., X.G., and X.T. designed the experiments. E.W., X.G., and X.T. performed main experiments and data analysis. R.Z., Y.C., D.Z., and Q.L. helped with ultracentrifugation and FISH experiments etc. F.L. helped with Apex techniques. All authors discussed and interpreted the results. E.W., X.G., X.T., J.L., and J.W. prepared the manuscript with input from other authors.

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Correspondence to Jianjun Luo, Jiangyun Wang or Runsheng Chen.

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Wu, E., Guo, X., Teng, X. et al. Discovery of Plasma Membrane-Associated RNAs through APEX-seq. Cell Biochem Biophys 79, 905–917 (2021). https://doi.org/10.1007/s12013-021-00991-0

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