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A high-throughput screen identifies the long non-coding RNA DRAIC as a regulator of autophagy

Abstract

Autophagy is a conserved degradation process that occurs in all eukaryotic cells and its dysfunction has been associated with various diseases including cancer. While a number of large-scale attempts have recently identified new molecular players in autophagy regulation, including proteins and microRNAs, little is known regarding the function of long non-coding RNAs (lncRNAs) in the regulation of this process. To identify new long non-coding RNAs with functional implications in autophagy, we performed a high-throughput RNAi screen targeting more than 600 lncRNA transcripts and monitored their effects on autophagy in MCF-7 cells. We identified 63 lncRNAs that affected GFP-LC3B puncta numbers significantly. We validated the strongest hit, the lncRNA DRAIC previously shown to impact cell proliferation, and revealed a novel role for this lncRNA in the regulation of autophagic flux. Interestingly, we find DRAIC’s pro-proliferative effects to be autophagy-independent. This study serves as a valuable resource for researchers from both the lncRNA and autophagy fields as it advances the current understanding of autophagy regulation by non-coding RNAs.

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References

  1. 1.

    Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.

  2. 2.

    Zhao YG, Zhang H. Formation and maturation of autophagosomes in higher eukaryotes: a social network. Curr Opin Cell Biol. 2018;53:29–36.

  3. 3.

    Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. (3rd edn) Autophagy. 2016;12:1–222.

  4. 4.

    Mizushima N, Levine B. Autophagy in mammalian development and differentiation. Nat Cell Biol. 2010;12:823–30.

  5. 5.

    Deretic V, Levine B. Autophagy, immunity, and microbial adaptations. Cell Host Microbe. 2009;5:527–49.

  6. 6.

    Fulda S, Kogel D. Cell death by autophagy: emerging molecular mechanisms and implications for cancer therapy. Oncogene. 2015;34:5105–13.

  7. 7.

    Choi AMK, Ryter SW, Levine B. Autophagy in human health and disease. New Engl J Med. 2013;368:651–62.

  8. 8.

    White E, Mehnert JM, Chan CS. Autophagy, metabolism, and cancer. Clin Cancer Res: Off J Am Assoc Cancer Res. 2015;21:5037–46.

  9. 9.

    van Beek N, Klionsky DJ, Reggiori F. Genetic aberrations in macroautophagy genes leading to diseases. Biochim Biophys Acta. 2018;1865:803–16.

  10. 10.

    Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol. 2009;10:458–67.

  11. 11.

    Abada A, Elazar Z. Getting ready for building: signaling and autophagosome biogenesis. EMBO Rep. 2014;15:839–52.

  12. 12.

    Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36:2503–18.

  13. 13.

    Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13:132–41.

  14. 14.

    Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell. 2009;20:1981–91.

  15. 15.

    Frankel LB, Lund AH. MicroRNA regulation of autophagy. Carcinogenesis. 2012;33:2018–25.

  16. 16.

    Quinn JJ, Chang HY. Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet. 2016;17:47–62.

  17. 17.

    Frankel LB, Lubas M, Lund AH. Emerging connections between RNA and autophagy. Autophagy. 2017;13:3–23.

  18. 18.

    Zhang J, Wang P, Wan L, Xu S, Pang D. The emergence of noncoding RNAs as Heracles in autophagy. Autophagy. 2017;13:1004–24.

  19. 19.

    Yang L, Wang H, Shen Q, Feng L, Jin H. Long non-coding RNAs involved in autophagy regulation. Cell Death Dis. 2017;8:e3073.

  20. 20.

    Xu Z, Yan Y, Qian L, Gong Z. Long non-coding RNAs act as regulators of cell autophagy in diseases (Review). Oncol Rep. 2017;37:1359–66.

  21. 21.

    Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014;505:344–52.

  22. 22.

    Wang K, Liu CY, Zhou LY, Wang JX, Wang M, Zhao B, et al. APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p. Nat Commun. 2015;6:6779.

  23. 23.

    YiRen H, YingCong Y, Sunwu Y, Keqin L, Xiaochun T, Senrui C, et al. Long noncoding RNA MALAT1 regulates autophagy associated chemoresistance via miR-23b-3p sequestration in gastric cancer. Mol Cancer. 2017;16:174.

  24. 24.

    Huang S, Lu W, Ge D, Meng N, Li Y, Su L, et al. A new microRNA signal pathway regulated by long noncoding RNA TGFB2-OT1 in autophagy and inflammation of vascular endothelial cells. Autophagy. 2015;11:2172–83.

  25. 25.

    Viereck J, Kumarswamy R, Foinquinos A, Xiao K, Avramopoulos P, Kunz M, et al. Long noncoding RNA Chast promotes cardiac remodeling. Sci Transl Med. 2016;8:326ra22.

  26. 26.

    Pawar K, Hanisch C, Palma Vera SE, Einspanier R, Sharbati S. Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy. Sci Rep. 2016;6:19416.

  27. 27.

    Ying L, Huang Y, Chen H, Wang Y, Xia L, Chen Y, et al. Downregulated MEG3 activates autophagy and increases cell proliferation in bladder cancer. Mol Biosyst. 2013;9:407–11.

  28. 28.

    Liu X, Xiao ZD, Han L, Zhang J, Lee SW, Wang W, et al. LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress. Nat Cell Biol. 2016;18:431–42.

  29. 29.

    Polycarpou-Schwarz M, Gross M, Mestdagh P, Schott J, Grund SE, Hildenbrand C. The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation. Oncogene. 2018;37:4750–68.

  30. 30.

    Notzold L, Frank L, Gandhi M, Polycarpou-Schwarz M, Gross M, Gunkel M, et al. The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells. Scientific reports. 2017;7.

  31. 31.

    Klingenberg M, Gross M, Goyal A, Polycarpou-Schwarz M, Miersch T, Ernst AS. The lncRNA CASC9 and RNA binding protein HNRNPL form a complex and co-regulate genes linked to AKT signaling. Hepatology. 2018;68:1817–32.

  32. 32.

    Seiler J, Breinig M, Caudron-Herger M, Polycarpou-Schwarz M, Boutros M, Diederichs S. The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance. Nucl Acids Res. 2017;45:5458–69.

  33. 33.

    Sakurai K, Reon BJ, Anaya J, Dutta A. The lncRNA DRAIC/PCAT29 Locus Constitutes a Tumor-Suppressive Nexus. Mol Cancer Res: MCR. 2015;13:828–38.

  34. 34.

    Sun M, Gadad SS, Kim DS, Kraus WL. Discovery, annotation, and functional analysis of long noncoding RNAs controlling cell-cycle gene expression and proliferation in breast cancer cells. Mol Cell. 2015;59:698–711.

  35. 35.

    Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007;25:193–205.

  36. 36.

    Konig R, Chiang CY, Tu BP, Yan SF, DeJesus PD, Romero A, et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods. 2007;4:847–9.

  37. 37.

    Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of transcription in human cells. Nature. 2012;489:101–8.

  38. 38.

    Zhao D, Dong JT. Upregulation of long non-coding RNA DRAIC correlates with adverse features of breast cancer. Non-coding RNA. 2018;4:39 https://doi.org/10.3390/ncrna4040039.

  39. 39.

    Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2:401–4.

  40. 40.

    Gómez-Sánchez R, Yakhine-Diop SMS, Rodríguez-Arribas M, Bravo-San Pedro JM, Martínez-Chacón G, Uribe-Carretero E, et al. mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines. Data Brief. 2016;7:641–7.

  41. 41.

    Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci. 2004;117(Pt 13):2805–12.

  42. 42.

    Lamark T, Kirkin V, Dikic I, Johansen T. NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle (Georget, Tex). 2009;8:1986–90.

  43. 43.

    Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol. 2005;171:603–14.

  44. 44.

    O’Prey J, Sakamaki J, Baudot AD, New M, Van Acker T, Tooze SA, et al. Application of CRISPR/Cas9 to autophagy research. Methods Enzymol. 2017;588:79–108.

  45. 45.

    Farkas T, Hoyer-Hansen M, Jaattela M. Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux. Autophagy. 2009;5:1018–25.

  46. 46.

    Hosokawa N, Hara Y, Mizushima N. Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett. 2006;580:2623–9.

  47. 47.

    Jung CH, Ro SH, Cao J, Otto NM, Kim DH. mTOR regulation of autophagy. FEBS Lett. 2010;584:1287–95.

  48. 48.

    Alers S, Loffler AS, Wesselborg S, Stork B. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012;32:2–11.

  49. 49.

    Kim D-H, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002;110:163–75.

  50. 50.

    Figueiredo VC, Markworth JF, Cameron-Smith D. Considerations on mTOR regulation at serine 2448: implications for muscle metabolism studies. Cell Mol Life Sci. 2017;74:2537–45.

  51. 51.

    Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102:15545–50.

  52. 52.

    el-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jackman J, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994;54:1169–74.

  53. 53.

    Mittnacht S. Control of pRB phosphorylation. Curr Opin Genet Dev. 1998;8:21–7.

  54. 54.

    Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem. 2008;283:1026–33.

  55. 55.

    Green DR, Levine B. To be or not to be? How selective autophagy and cell death govern cell fate. Cell. 2014;157:65–75.

  56. 56.

    Geisler S, Coller J. RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol. 2013;14:699–712.

  57. 57.

    Chen CL, Tseng YW, Wu JC, Chen GY, Lin KC, Hwang SM, et al. Suppression of hepatocellular carcinoma by baculovirus-mediated expression of long non-coding RNA PTENP1 and MicroRNA regulation. Biomaterials. 2015;44:71–81.

  58. 58.

    Ge D, Han L, Huang S, Peng N, Wang P, Jiang Z, et al. Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells. Autophagy. 2014;10:957–71.

  59. 59.

    Song J, Ahn C, Chun CH, Jin EJ. A long non-coding RNA, GAS5, plays a critical role in the regulation of miR-21 during osteoarthritis. J Orthop Res: Off Publ Orthop Res Soc. 2014;32:1628–35.

  60. 60.

    Huang F, Chen W, Peng J, Li Y, Zhuang Y, Zhu Z, et al. LncRNA PVT1 triggers Cyto-protective autophagy and promotes pancreatic ductal adenocarcinoma development via the miR-20a-5p/ULK1 Axis. Mol Cancer. 2018;17:98.

  61. 61.

    Yang L, Zhang X, Li H, Liu J. The long noncoding RNA HOTAIR activates autophagy by upregulating ATG3 and ATG7 in hepatocellular carcinoma. Mol Biosyst. 2016;12:2605–12.

  62. 62.

    Chen YN, Cai MY, Xu S, Meng M, Ren X, Yang JW, et al. Identification of the lncRNA, AK156230, as a novel regulator of cellular senescence in mouse embryonic fibroblasts. Oncotarget. 2016;7:52673–84.

  63. 63.

    Wang Y, Guo Q, Zhao Y, Chen J, Wang S, Hu J, et al. BRAF-activated long non-coding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma. Oncol Lett. 2014;8:1947–52.

  64. 64.

    Guo S, Pridham KJ, Virbasius C-M, He B, Zhang L, Varmark H, et al. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages. Sci Rep. 2018;8:2822.

  65. 65.

    Frankel LB, Wen J, Lees M, Hoyer-Hansen M, Farkas T, Krogh A, et al. microRNA-101 is a potent inhibitor of autophagy. EMBO J. 2011;30:4628–41.

  66. 66.

    Lipinski MM, Hoffman G, Ng A, Zhou W, Py BF, Hsu E, et al. A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions. Dev Cell. 2010;18:1041–52.

  67. 67.

    Liu C, Zhang Y, She X, Fan L, Li P, Feng J, et al. A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy. J Hematol Oncol. 2018;11:77.

  68. 68.

    Lubas M, Harder LM, Kumsta C, Tiessen I, Hansen M, Andersen JS. eIF5A is required for autophagy by mediating ATG3 translation. EMBO Rep.2018;19:e46072 https://doi.org/10.15252/embr.201846072.

  69. 69.

    McKnight NC, Jefferies HB, Alemu EA, Saunders RE, Howell M, Johansen T, et al. Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC. EMBO J. 2012;31:1931–46.

  70. 70.

    Orvedahl A, Sumpter R Jr., Xiao G, Ng A, Zou Z, Tang Y, et al. Image-based genome-wide siRNA screen identifies selective autophagy factors. Nature. 2011;480:113–7.

  71. 71.

    Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Muller M, et al. Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc Natl Acad Sci USA. 2005;102:7922–7.

  72. 72.

    Litovchick L, Sadasivam S, Florens L, Zhu X, Swanson SK, Velmurugan S, et al. Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence. Mol Cell. 2007;26:539–51.

  73. 73.

    Loayza-Puch F, Drost J, Rooijers K, Lopes R, Elkon R, Agami R. p53 induces transcriptional and translational programs to suppress cell proliferation and growth. Genome Biol. 2013;14:R32.

  74. 74.

    Musiwaro P, Smith M, Manifava M, Walker SA, Ktistakis NT. Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy. 2013;9:1407–17.

  75. 75.

    Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol. 2013;14:518–28.

  76. 76.

    Capparelli C, Chiavarina B, Whitaker-Menezes D, Pestell TG, Pestell RG, Hulit J, et al. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis. Cell Cycle (Georget, Tex). 2012;11:3599–610.

  77. 77.

    Jiang H, Martin V, Gomez-Manzano C, Johnson DG, Alonso M, White E, et al. The RB-E2F1 pathway regulates autophagy. Cancer Res. 2010;70:7882–93.

  78. 78.

    Boutros M, Ahringer J. The art and design of genetic screens: RNA interference. Nat Rev Genet. 2008;9:554–66.

  79. 79.

    Poulsen EG, Nielsen SV, Pietras EJ, Johansen JV, Steinhauer C, Hartmann-Petersen R. High-throughput siRNA screening applied to the ubiquitin-proteasome system. Methods Mol Biol (Clifton, NJ). 2016;1449:421–39.

  80. 80.

    Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–U54.

  81. 81.

    Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

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Acknowledgements

We thank Sudeep Sahadevan for analysis of RNA-seq data, Bettina Mentz for technical assistance, Kevin Ryan for providing pLenti-CRISPR NTC, ATG5, and ATG7 constructs [44], W Lee Kraus for providing PInducer20-DRAIC and -EV constructs [34]. Imke Tiessen was supported by the People Program (Marie Skłodowska-Curie Actions) of the European Union’s Seventh Framework Program FP7-PEOPLE-2013-ITN (Grant agreement no.: 607720). Work in the Lund laboratory was supported by the Danish Council for Independent Research (Sapere Aude program), the Novo Nordisk Foundation, the Lundbeck Foundation, the Danish Cancer Society and the EU COST Action Transautophagy (CA15138).

Author information

IT, ML, LBF, and AHL designed and interpreted the experiments. IT, LBF, and AHL wrote the manuscript. IT, MHA, and LBF performed experiments. CS and EJP assisted with screening and automated quantifications. HMG performed the GSEA analysis. SD provided the siRNA library and commented the manuscript.

Correspondence to Lisa B. Frankel or Anders H. Lund.

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