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3′-Terminal 2′-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute 2

Liang, Hongwei, Jiao, Zichen, Rong, Weiwei, Qu, Shuang, Liao, Zhicong, Sun, Xinlei, Wei, Yao, Zhao, Quan, Wang, Jun, Liu, Yuan, Chen, Xi, Wang, Tao, Zhang, Chen-Yu, Zen, Ke·2020·Nucleic Acids Research
DOI 10.1093/nar/gkaa504PMC PMC7367198
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Abstract

Methylation of miRNAs at the 2′-hydroxyl group on the ribose at 3′-end (2′-O-methylation, 2′Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC–MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3′-terminal 2′Ome. Predominant 3′-terminal 2′Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/β-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3′-terminal 2′Ome. We further identify HENMT1 as the methyltransferase responsible for 3′-terminal 2′Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3′→5′ exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3′-terminal 2′Ome of mammalian miRNAs and highlight its role in enhancing miRNA’s stability and function.

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Figure 1. Detection of 2′Ome of human miRNAs at the 3′-end. (A) LC−MS/MS chromatogram in MRM mode of the synthetic nucleosides (standard, std) and authentic nucleosides isolated from non-cancerous lung tissue (N) and NSCLC lung tissue (C). (B) Comparison of nucleoside methylation in the indicated small RNAs (18–24nt) from non-cancerous lung tissue (N) and NSCLC lung tissue (C). (C) Heatmap of 2′Ome at the 3′-end of human miRNAs in non-cancerous lung tissue (N) and NSCLC lung tissue (C) after oxidation procedure. N-oxid: non-cancerous lung tissue with oxidation. C-oxid: NSCLC lung tissue with oxidation. (D) Detection of 2′Ome of human miRNAs at the 3′-end by qRT-PCR after oxidation procedure. (E) Detection of 2′Ome of human miRNAs at the 3′-end by northern blotting after Oxid/β-elim procedure.
Figure 1. Detection of 2′Ome of human miRNAs at the 3′-end. (A) LC−MS/MS chromatogram in MRM mode of the synthetic nucleosides (standard, std) and authentic nucleosides isolated from non-cancerous lung tissue (N) and NSCLC lung tissue (C). (B) Comparison of nucleoside methylation in the indicated small RNAs (18–24nt) from non-cancerous lung tissue (N) and NSCLC lung tissue (C). (C) Heatmap of 2′Ome at the 3′-end of human miRNAs in non-cancerous lung tissue (N) and NSCLC lung tissue (C) after oxidation procedure. N-oxid: non-cancerous lung tissue with oxidation. C-oxid: NSCLC lung tissue with oxidation. (D) Detection of 2′Ome of human miRNAs at the 3′-end by qRT-PCR after oxidation procedure. (E) Detection of 2′Ome of human miRNAs at the 3′-end by northern blotting after Oxid/β-elim procedure.CC BY-NC · PubMed Central
Figure 2. Detection of 3′-terminal 2′Ome of miR-21-5p and miR-26-5p in NSCLC and non-cancerous lung tissues. (A) Left, depict of experimental strategy for isolating miR-21-5p and miR-26-5p from NSCLC and non-cancerous lung tissues. Right, purity of isolated miR-21-5p or miR-26-5p examined by Solexa sequencing. (B, C) Detection of 3′-terminal 2′Ome of miR-21-5p or miR-26-5p in NSCLC (C) and non-cancerous lung tissues (N) by Mass spectrometry (B) and LC–MS/MS (C).
Figure 2. Detection of 3′-terminal 2′Ome of miR-21-5p and miR-26-5p in NSCLC and non-cancerous lung tissues. (A) Left, depict of experimental strategy for isolating miR-21-5p and miR-26-5p from NSCLC and non-cancerous lung tissues. Right, purity of isolated miR-21-5p or miR-26-5p examined by Solexa sequencing. (B, C) Detection of 3′-terminal 2′Ome of miR-21-5p or miR-26-5p in NSCLC (C) and non-cancerous lung tissues (N) by Mass spectrometry (B) and LC–MS/MS (C).CC BY-NC · PubMed Central
Figure 3. Level of methylated miR-21-5p in sera from NSCLC patients and healthy donors. (A) Elevation of methylated miR-21-5p level in serum samples from NSCLC patients (n = 21) than that from healthy donors (n = 11). (B) Decrease of methylated miR-21-5p level in serum samples from NSCLC patients (n = 20) before and after tumor removal by surgery. **P < 0.01, ***P < 0.001.
Figure 3. Level of methylated miR-21-5p in sera from NSCLC patients and healthy donors. (A) Elevation of methylated miR-21-5p level in serum samples from NSCLC patients (n = 21) than that from healthy donors (n = 11). (B) Decrease of methylated miR-21-5p level in serum samples from NSCLC patients (n = 20) before and after tumor removal by surgery. **P < 0.01, ***P < 0.001.CC BY-NC · PubMed Central
Figure 4. HEMT1-knockout impairs 3′-terminal 2′Ome of miRNAs in A549 cells and mice. (A) HENMT1 expression in human tissues. (B) HENMT1 expression and location in A549 cell. Left: localization of HENMT1 detected by immunofluorescence analysis; right: localization of HENMT1 detected by Western blot. (C) HENMT1 expression detected by Western blot in WT A549 cells (WT), scramble gDNA (control cells) and HEMT1-knockout A549 cells (HEMT1 Knockout) via Crisper/cas9 technique. (D–F) Levels of miRNAs in A549 or HENMT1-knockout A549 (A549KO) cells detected by high-through sequencing after oxidation (D), qRT-PCR after oxidation (E) and Northern blot (F) after Oxid/β-elim procedure. (G) HENMT1 expression detected by western blot in lung derived from wild type mice (WT) and HEMT1-knockout mice (KO). (H) Ratio of piR_020485, miR-21-5p and miR-26a-5p levels with oxidation versus piR_020485, miR-21-5p and miR-26a-5p levels without oxidation in lung tissues from WT and HENMT1-KO (KO) mice by qRT-PCR. (I) Northern blot analysis of piR_020485 and miR-26a-5p expression in lung tissues from WT and KO mice after Oxid/β-elim procedure.
Figure 4. HEMT1-knockout impairs 3′-terminal 2′Ome of miRNAs in A549 cells and mice. (A) HENMT1 expression in human tissues. (B) HENMT1 expression and location in A549 cell. Left: localization of HENMT1 detected by immunofluorescence analysis; right: localization of HENMT1 detected by Western blot. (C) HENMT1 expression detected by Western blot in WT A549 cells (WT), scramble gDNA (control cells) and HEMT1-knockout A549 cells (HEMT1 Knockout) via Crisper/cas9 technique. (D–F) Levels of miRNAs in A549 or HENMT1-knockout A549 (A549KO) cells detected by high-through sequencing after oxidation (D), qRT-PCR after oxidation (E) and Northern blot (F) after Oxid/β-elim procedure. (G) HENMT1 expression detected by western blot in lung derived from wild type mice (WT) and HEMT1-knockout mice (KO). (H) Ratio of piR_020485, miR-21-5p and miR-26a-5p levels with oxidation versus piR_020485, miR-21-5p and miR-26a-5p levels without oxidation in lung tissues from WT and HENMT1-KO (KO) mice by qRT-PCR. (I) Northern blot analysis of piR_020485 and miR-26a-5p expression in lung tissues from WT and KO mice after Oxid/β-elim procedure.CC BY-NC · PubMed Central
Figure 5. In vitro assay of methylation of synthetic miR-21-5p by HEMT1. The methylation assay is carried out in 20 μl reaction buffer containing 10 μM synthetic miR-21-5p and 5μM HENMT1 recombinant proteins. After incubating at 37°C for 40 min, RNA was recovered and purified by ethanol precipitation. (A–E) Methylation of synthetic miR-21-5p at 3′-end by purified HENMT1 assessed by EPIgeneous™ methyltransferase assay (A), Northern blot (B), qRT-PCR (C), mass spectrometry (D) and LC–MS/MS (E), respectively.
Figure 5. In vitro assay of methylation of synthetic miR-21-5p by HEMT1. The methylation assay is carried out in 20 μl reaction buffer containing 10 μM synthetic miR-21-5p and 5μM HENMT1 recombinant proteins. After incubating at 37°C for 40 min, RNA was recovered and purified by ethanol precipitation. (A–E) Methylation of synthetic miR-21-5p at 3′-end by purified HENMT1 assessed by EPIgeneous™ methyltransferase assay (A), Northern blot (B), qRT-PCR (C), mass spectrometry (D) and LC–MS/MS (E), respectively.CC BY-NC · PubMed Central
Figure 6. 3′-terminal 2′Ome of human miR-21-5p increases its stability. (A) The half-life of miR-21-5p and miR-26-5p in A549 cells and A549KO (HENMT1-knockout) cells. (B, C) Direct degradation of synthetic miR-21-5p/miR-21-5pCH3 and miR-26-5p/miR-26-5pCH3 by PNPT1 recombination protein in a time-dependent (B) and concentration-dependent (C) manner.
Figure 6. 3′-terminal 2′Ome of human miR-21-5p increases its stability. (A) The half-life of miR-21-5p and miR-26-5p in A549 cells and A549KO (HENMT1-knockout) cells. (B, C) Direct degradation of synthetic miR-21-5p/miR-21-5pCH3 and miR-26-5p/miR-26-5pCH3 by PNPT1 recombination protein in a time-dependent (B) and concentration-dependent (C) manner.CC BY-NC · PubMed Central
Figure 7. 3′-Terminal 2′Ome of human miR-21-5p increases its association with AGO2 complex and inhibition on PDCD4 expression. (A) Depict of experimental strategy of assaying binding affinity of miR-21-5p or miR-21-5pCH3 with AGO2 complex using Cy3- or Cy5-labeled miRNAs. (B) Binding affinity of miR-21-5p and miR-21-5pCH3 with AGO2 complex detected by fluorescence intensity measurement. (C) Kinetics of binding of miR-21-5p and miR-21-5pCH3 with AGO2 detected by microscale thermophoresis. (D) Binding of miR-21-5p or miR-26-5p with AGO2 detected by the luciferase activity in A549 or A549KO cells transfected with miR-21-5p or miR-26-5p reporter plasmid. (E) PDCD4 and ULK1 protein level in A549 and A549KO cells at different time points after serum depletion. (F) Apoptosis of A549 and A549KO cells induced by serum depletion.
Figure 7. 3′-Terminal 2′Ome of human miR-21-5p increases its association with AGO2 complex and inhibition on PDCD4 expression. (A) Depict of experimental strategy of assaying binding affinity of miR-21-5p or miR-21-5pCH3 with AGO2 complex using Cy3- or Cy5-labeled miRNAs. (B) Binding affinity of miR-21-5p and miR-21-5pCH3 with AGO2 complex detected by fluorescence intensity measurement. (C) Kinetics of binding of miR-21-5p and miR-21-5pCH3 with AGO2 detected by microscale thermophoresis. (D) Binding of miR-21-5p or miR-26-5p with AGO2 detected by the luciferase activity in A549 or A549KO cells transfected with miR-21-5p or miR-26-5p reporter plasmid. (E) PDCD4 and ULK1 protein level in A549 and A549KO cells at different time points after serum depletion. (F) Apoptosis of A549 and A549KO cells induced by serum depletion.CC BY-NC · PubMed Central

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Research works printed but not linked: 34

  1. Chen, 2008, Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases, Cell Res., 18, 997, 10.1038/cr.2008.282 · DOI · Paper page
  2. Chen, 2012, Secreted microRNAs: a new form of intercellular communication, Trends Cell Biol., 22, 125, 10.1016/j.tcb.2011.12.001 · DOI · Paper page
  3. Zen, 2012, Circulating microRNAs: a novel class of biomarkers to diagnose and monitor human cancers, Med. Res. Rev., 32, 326, 10.1002/med.20215 · DOI
  4. Chen, 2012, Horizontal transfer of microRNAs: molecular mechanisms and clinical applications, Protein Cell, 3, 28, 10.1007/s13238-012-2003-z · DOI
  5. Cortez, 2011, MicroRNAs in body fluids–the mix of hormones and biomarkers, Nat. Rev. Clin. Oncol., 8, 467, 10.1038/nrclinonc.2011.76 · DOI
  6. Mori, 2019, Extracellular miRNAs: from biomarkers to mediators of physiology and disease, Cell Metab., 30, 656, 10.1016/j.cmet.2019.07.011 · DOI
  7. Turchinovich, 2016, Extracellular miRNA: a collision of two paradigms, Trends Biochem. Sci., 41, 883, 10.1016/j.tibs.2016.08.004 · DOI
  8. Quinn, 2015, Extracellular RNAs: development as biomarkers of human disease, J. Extracell. Vesicles, 4, 10.3402/jev.v4.27495 · DOI
  9. Li, 2012, Argonaute 2 complexes selectively protect the circulating microRNAs in cell-secreted microvesicles, PLoS One, 7, e46957, 10.1371/journal.pone.0046957 · DOI
  10. Yu, 2005, Methylation as a crucial step in plant microRNA biogenesis, Science, 307, 932, 10.1126/science.1107130 · DOI
  11. Abe, 2014, Impact of age-associated increase in 2′-O-methylation of miRNAs on aging and neurodegeneration in Drosophila, Genes Dev., 28, 44, 10.1101/gad.226654.113 · DOI · Paper page
  12. Hoerter, 2007, Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum, RNA, 13, 1887, 10.1261/rna.602307 · DOI
  13. Kirino, 2007, Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini, Nat. Struct. Mol. Biol., 14, 347, 10.1038/nsmb1218 · DOI
  14. Ji, 2012, Regulation of small RNA stability: methylation and beyond, Cell Res., 22, 624, 10.1038/cr.2012.36 · DOI
  15. Lim, 2015, HENMT1 and piRNA stability are required for adult male germ cell transposon repression and to define the spermatogenic program in the mouse, PLos Genet., 11, e1005782, 10.1371/journal.pgen.1005782 · DOI
  16. Alberti, 2018, Cell-type specific sequencing of microRNAs from complex animal tissues, Nat. Methods, 15, 283, 10.1038/nmeth.4610 · DOI
  17. Ohara, 2007, The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated, Nat. Struct. Mol. Biol., 14, 349, 10.1038/nsmb1220 · DOI
  18. Wei, 2014, Importin 8 regulates the transport of mature MicroRNAs into the cell nucleus, J. Biol. Chem., 289, 10270, 10.1074/jbc.C113.541417 · DOI
  19. Wang, 2018, Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR, RNA, 24, 1520, 10.1261/rna.065144.117 · DOI
  20. Cheng, 2015, miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn's disease, Sci. Rep., 5, 10397, 10.1038/srep10397 · DOI
  21. Chan, 2009, Structural and biochemical insights into 2′-O-methylation at the 3′-terminal nucleotide of RNA by Hen1, Proc. Natl Acad. Sci. U.S.A., 106, 17699, 10.1073/pnas.0907540106 · DOI
  22. Yan, 2013, A high-throughput quantitative approach reveals more small RNA modifications in mouse liver and their correlation with diabetes, Anal. Chem., 85, 12173, 10.1021/ac4036026 · DOI
  23. Kumarswamy, 2011, Regulation and function of miRNA-21 in health and disease, RNA Biol, 8, 706, 10.4161/rna.8.5.16154 · DOI
  24. Kirino, 2007, The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs, RNA, 13, 1397, 10.1261/rna.659307 · DOI
  25. Mickute, 2018, Animal Hen1 2′-O-methyltransferases as tools for 3′-terminal functionalization and labelling of single-stranded RNAs, Nucleic Acids Res., 46, e104, 10.1093/nar/gky514 · DOI
  26. Kimos, 2016, Development of an HTRF assay for the detection and characterization of inhibitors of catechol-O-methyltransferase, J. Biomol. Screen, 21, 490, 10.1177/1087057115616793 · DOI
  27. Ruegger, 2012, MicroRNA turnover: when, how, and why, Trends Biochem. Sci., 37, 436, 10.1016/j.tibs.2012.07.002 · DOI
  28. Kuhn, 2013, Eukaryotic Argonautes come into focus, Trends Biochem. Sci., 38, 263, 10.1016/j.tibs.2013.02.008 · DOI
  29. Elkayam, 2012, The structure of human argonaute-2 in complex with miR-20a, Cell, 150, 100, 10.1016/j.cell.2012.05.017 · DOI
  30. Jin, 2017, MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy, Cell Death. Dis., 8, e2540, 10.1038/cddis.2016.461 · DOI
  31. Zhang, 2012, MicroRNA degradation and turnover: regulating the regulators, Wiley Interdiscip. Rev. RNA, 3, 593, 10.1002/wrna.1114 · DOI
  32. Ameres, 2010, Target RNA-directed trimming and tailing of small silencing RNAs, Science, 328, 1534, 10.1126/science.1187058 · DOI · Paper page
  33. Ren, 2014, Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage, Proc. Natl Acad. Sci. U.S.A., 111, 6365, 10.1073/pnas.1405083111 · DOI
  34. Ren, 2014, Small RNAs meet their targets: when methylation defends miRNAs from uridylation, RNA Biol., 11, 1099, 10.4161/rna.36243 · DOI

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Metadata

Authors
Liang, H., Jiao, Z., Rong, W., Qu, S., Liao, Z., Sun, X., Wei, Y., Zhao, Q., Wang, J., Liu, Y., Chen, X., Wang, T., Zhang, C.-Y., & Zen, K.
Affiliations
  • State Key Laboratory of Pharmaceutical Biotechnology, Department of Gastroenterology, Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210093, China
  • Department of Cardiothoracic Surgery, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
  • Department of Emergency Medicine, Nanjing Drum Tower Hospital, Medical School, Nanjing University, 210008 Nanjing, China
  • Center for Inflammation, Immunity and Infectious Diseases, Georgia State University, Atlanta, GA 30032, USA
ORCID
Zen, Ke
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2020-06-15
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkaa504
PMC
PMC7367198
Cited by
21
References
2
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • Ministry of Science and Technology (2018YFA0507100)
  • National Natural Science Foundation of China (31801088)
  • Natural Science Foundation of Jiangsu Province (BK20170076)
  • China Postdoctoral Science Foundation (2018M642210)
  • Jiangsu Province Postdoctoral Fund (2018K050A)

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