Small RNA修饰芯片

• 簡介
• 挑戰及解決方案
• 數據庫
• 結果展示

• Arraystar small RNA修饰芯片技术服务在单张芯片可定量miRNA，pre-miRNA和tRNA衍生的small RNA（tsRNA，包括tRF＆tiRNA）的碱基修饰。可检测的修饰包括：8-氧代鸟嘌呤（o8G），7-甲基鸟苷（m7G），N6-甲基腺苷（m6A），假尿苷（Ψ）、5-甲基胞苷（m5C）或 N1-甲基腺苷(m1A)。

  
  

  芯片優點

  • 能够检测及定量多种small RNA上修饰：包括o8G，m7G，m6A，Ψ，m5C或m1A

  • 能够检测多种small RNA：包括miRNA，pre-miRNA和tsRNAs（tRF＆tiRNA）

  • 金标准准确定量small RNA的修饰: 直接RNA末端标记，避免了测序建库过程中因修饰导致的cDNA延伸中止的情况，可确保对small RNA修饰定量的高保真性。

  • 高灵敏度检测低水平small RNA的修饰：克服二代測序的侷限性，对低表达或低修饰水平的small RNA分析具有出色的分析灵敏度。

  • 所需样品量少，总RNA量可低至2 µg。

  
  

  Arraystar Small RNA修饰芯片列表

  服务名称	可检测的修饰*	描述	规格
  Arraystar Human Small RNA 修饰芯片	O8G/m7G/m6A/Ψ/m5C/m1A	定量miRNA，pre-miRNA, & tsRNA修饰	8 x 15K
  Arraystar Mouse Small RNA 修饰芯片	O8G/m7G/m6A/Ψ/m5C/m1A	定量miRNA，pre-miRNA, & tsRNA修饰	8 x 15K

  * 單張芯片可從6種修飾中選擇一種進行檢測。

  
  

  
  

• Small RNA修饰高通量筛选面临的挑战及解决方案

         尽管测序已用于small RNA高通量筛选，但RNA修饰对测序定量的影响仍被严重忽视。RNA上多种修饰（m1A，m3C和m1G等）会干扰测序建库过程中的逆转录，因此small RNA-seq对small RNA修饰的定量是不准确的，特别是对small RNA上的修饰。 例如，Small RNA-seq大多偏向检测18nt的3’tsRNA，而Northern blot主要检测到的是22nt的同工型3’tsRNA。这是由于TUC存在m1A，会抑制逆转录酶进行逆转录。大多数small RNA测序数据是从上述文库构建方法中获得的，因此，对于有修饰的small RNA，这些数据可能产生误导。

         同样，small RNA-seq需要多个PCR扩增步骤，这会导致明显的定量偏差及不准确，因此需要使用独立正交方法。

         事实上，研究修饰的测序方法需要大量的样本（总RNA> 100 ug），这样对样本量有限的研究会产生极大的限制。

         此外，small RNA测序通常使用Reads Per Million（RPM）进行标准化，来表示样品中RNA的相对丰度。 然而，RPM取决于样品中small RNA的组成。 一个small RNA的RPM的变化将影响所有其它small RNA的值，即使它们的绝对表达水平没有改变。

         因此，就需要克服基于测序方法的局限，开发非测序技术，以更高的灵敏度和准确性来鉴定和定量small RNA的修饰谱。

  
  

  定量small RNA转录后修饰的技术

  Arraystar small RNA修饰芯片技术（图1）将small RNA芯片与RNA免疫沉淀（RIP）进行整合，可在一张芯片上同时检测修饰及未修饰small RNA水平，为修饰对small RNA（包括miRNA，pre-miRNA和tRF＆tiRNA）的调控提供重要信息，

  

  图1. Arraystar small RNA修饰芯片技术，分别鉴定和定量small RNA转录后修饰，分别为o8G，m7G，m6A，Ψ和m5C。使用特异性抗体通过免疫沉淀富集修饰的small RNA后，使用Arraystar small RNA修饰芯片进行鉴定和定量。

  
  

  
  

  參考文獻

  1.    Guzzi N et al: Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells. Cell 2018, 173(5):1204-1216 e1226.[PMID: 29628141]

  2.    Keam SP et al: The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Res 2014, 42(14):8984-8995.[PMID: 25038252]

  3.    Keam SP, Sobala A, Ten Have S, Hutvagner G: tRNA-Derived RNA Fragments Associate with Human Multisynthetase Complex (MSC) and Modulate Ribosomal Protein Translation. J Proteome Res 2017, 16(2):413-420.[PMID: 27936807]

  4.    Zhang X et al: IL-4 Inhibits the Biogenesis of an Epigenetically Suppressive PIWI-Interacting RNA To Upregulate CD1a Molecules on Monocytes/Dendritic Cells. J Immunol 2016, 196(4):1591-1603.[PMID: 26755820]

  5.    Honda S et al: The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells. Nucleic Acids Res 2017, 45(15):9108-9120.[PMID: 28645172]

  6.    Cole C et al: Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 2009, 15(12):2147-2160.[PMID: 19850906]

  7.    Sobala A, Hutvagner G: Small RNAs derived from the 5' end of tRNA can inhibit protein translation in human cells. RNA Biol 2013, 10(4):553-563.[PMID: 23563448]

  8.    Lee YS, Shibata Y, Malhotra A, Dutta A: A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 2009, 23(22):2639-2649.[PMID: 19933153]

  9.    Huang B et al: tRF/miR-1280 Suppresses Stem Cell-like Cells and Metastasis in Colorectal Cancer. Cancer Res 2017, 77(12):3194-3206.[PMID: 28446464]

  10.  Kuscu C et al: tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner. RNA 2018, 24(8):1093-1105.[PMID: 29844106]

  11.  Kim HK et al: A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature 2017, 552(7683):57-62.[PMID: 29186115]

  12.  Kim HK et al: A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice. Cell Rep 2019, 29(12):3816-3824 e3814.[PMID: 31851915]

  13.  Yeung ML et al: Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Res 2009, 37(19):6575-6586.[PMID: 19729508]

  14.  Schorn AJ, Gutbrod MJ, LeBlanc C, Martienssen R: LTR-Retrotransposon Control by tRNA-Derived Small RNAs. Cell 2017, 170(1):61-71 e11.[PMID: 28666125]

  15.  Maute RL et al: tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci U S A 2013, 110(4):1404-1409.[PMID: 23297232]

  16.  Ruggero K et al: Small noncoding RNAs in cells transformed by human T-cell leukemia virus type 1: a role for a tRNA fragment as a primer for reverse transcriptase. J Virol 2014, 88(7):3612-3622.[PMID: 24403582]

  17.  Falconi M et al: A novel 3'-tRNA(Glu)-derived fragment acts as a tumor-suppressor in breast cancer by targeting nucleolin. FASEB J 2019:fj201900382RR.[PMID: 31560576]

  18.  Zhou K et al: A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells. Oncotarget 2017, 8(56):95377-95391.[PMID: 29221134]

  19.  Goodarzi H et al: Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015, 161(4):790-802.[PMID: 25957686]

  20.  Natt D et al: Human sperm displays rapid responses to diet. PLoS Biol 2019, 17(12):e3000559.[PMID: 31877125]

  21.  Veneziano D et al: Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2019, 116(48):24252-24258.[PMID: 31723042]

  22.  Haussecker D et al: Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 2010, 16(4):673-695.[PMID: 20181738]

  23.  Balatti V et al: tsRNA signatures in cancer. Proc Natl Acad Sci U S A 2017, 114(30):8071-8076.[PMID: 28696308]

  24.  Cho H et al: Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3' trailer-derived tRNA fragments. Nucleic Acids Res 2019, 47(18):9888-9901.[PMID: 31504775]

  25.  Babiarz JE et al: Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev 2008, 22(20):2773-2785.[PMID: 18923076]

  26.  Hasler D et al: The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway. Mol Cell 2016, 63(1):110-124.[PMID: 27345152]

  27.  Pekarsky Y et al: Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer. Proc Natl Acad Sci U S A 2016, 113(18):5071-5076.[PMID: 27071132]

  28.  Liao JY et al: Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3' trailers. PLoS One 2010, 5(5):e10563.[PMID: 20498841]

  29.  La Ferlita A et al: Identification of tRNA-derived ncRNAs in TCGA and NCI-60 panel cell lines and development of the public database tRFexplorer. Database (Oxford) 2019, 2019.[PMID: 31735953]

  30.  Honda S et al: Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci U S A 2015, 112(29):E3816-3825.[PMID: 26124144]

  31.  Donovan J, Rath S, Kolet-Mandrikov D, Korennykh A: Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery. RNA 2017, 23(11):1660-1671.[PMID: 28808124]

  32.  Hanada T et al: CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 2013, 495(7442):474-480.[PMID: 23474986]

  33.  Saikia M et al: Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress. Mol Cell Biol 2014, 34(13):2450-2463.[PMID: 24752898]

  34.  Wang Q et al: Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Mol Ther 2013, 21(2):368-379.[PMID: 23183536]

  35.  Deng J et al: Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism. Mol Ther 2015, 23(10):1622-1629.[PMID: 26156244]

  36.  Zhou J et al: Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection. J Gen Virol 2017, 98(7):1600-1610.[PMID: 28708049]

  37.  Yang X et al: 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 2017, 27(5):606-625.[PMID: 28418038]

  38.  Ivanov P et al: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011, 43(4):613-623.[PMID: 21855800]

  39.  Ivanov P et al: G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments. Proc Natl Acad Sci U S A 2014, 111(51):18201-18206.[PMID: 25404306]

  40.  Schaffer AE et al: CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 2014, 157(3):651-663.[PMID: 24766810]

• Arraystar  Human small RNA 修饰芯片 V1.0

  探针总数	14,706
  探针设计策略	整个探针由5’cap区， small RNA特异性区和3’linker区组成。
  探针结合位点	5-p-miRNA 和 5'tsRNA: small RNA的3’区域 3-p-miRNA 和 3'tsRNA: small RNA的5’区域 Pre-miRNA: pre-miRNA的颈环区域设计
  探针特异性	small RNA特异性
  miRNA数目	2,628 (1,319个5-p-miRNAs, 1,309个3-p-miRNAs)
  pre-miRNAs数目	1,745
  tsRNAs数目	5,128
  Small RNA来源数据库	miRNA: miRBase (v22) pre-miRNA: miRBase (v22) tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08) 文獻: 公开发表的文献至 2019 [1-40]
  芯片规格	8 x 15K

  
  

  Arraystar Mouse small RNA 修饰芯片 V1.0

  探针总数	14,895
  探针设计策略	整个探针由5’cap区， small RNA特异性区和3’linker区组成。
  探针结合位点	5-p-miRNA 和 5'tsRNA: small RNA的3’区域 3-p-miRNA 和 3'tsRNA: small RNA的5’区域 Pre-miRNA: pre-miRNA的颈环区域设计
  探针特异性	small RNA特异性
  miRNA数目	1949 (966个5-p-miRNAs, 983个3-p-miRNAs)
  pre-miRNAs数目	1,122
  tsRNAs数目	1,809
  Small RNA来源数据库	miRNA: miRBase (v22) pre-miRNA: miRBase (v22) tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08) 文獻: 公开发表的文献至 2019 [1-40]
  芯片规格	8 x 15K

  
  

  References

  
  

  1. Guzzi N et al: Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells. Cell 2018, 173(5):1204-1216 e1226.[PMID: 29628141]

  2. Keam SP et al: The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Res 2014, 42(14):8984-8995.[PMID: 25038252]

  3. Keam SP, Sobala A, Ten Have S, Hutvagner G: tRNA-Derived RNA Fragments Associate with Human Multisynthetase Complex (MSC) and Modulate Ribosomal Protein Translation. J Proteome Res 2017, 16(2):413-420.[PMID: 27936807]

  4. Zhang X et al: IL-4 Inhibits the Biogenesis of an Epigenetically Suppressive PIWI-Interacting RNA To Upregulate CD1a Molecules on Monocytes/Dendritic Cells. J Immunol 2016, 196(4):1591-1603.[PMID: 26755820]

  5. Honda S et al: The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells. Nucleic Acids Res 2017, 45(15):9108-9120.[PMID: 28645172]

  6. Cole C et al: Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 2009, 15(12):2147-2160.[PMID: 19850906]

  7. Sobala A, Hutvagner G: Small RNAs derived from the 5' end of tRNA can inhibit protein translation in human cells. RNA Biol 2013, 10(4):553-563.[PMID: 23563448]

  8. Lee YS, Shibata Y, Malhotra A, Dutta A: A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 2009, 23(22):2639-2649.[PMID: 19933153]

  9. Huang B et al: tRF/miR-1280 Suppresses Stem Cell-like Cells and Metastasis in Colorectal Cancer. Cancer Res 2017, 77(12):3194-3206.[PMID: 28446464]

  10. Kuscu C et al: tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner. RNA 2018, 24(8):1093-1105.[PMID: 29844106]

  11. Kim HK et al: A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature 2017, 552(7683):57-62.[PMID: 29186115]

  12. Kim HK et al: A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice. Cell Rep 2019, 29(12):3816-3824 e3814.[PMID: 31851915]

  13. Yeung ML et al: Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Res 2009, 37(19):6575-6586.[PMID: 19729508]

  14. Schorn AJ, Gutbrod MJ, LeBlanc C, Martienssen R: LTR-Retrotransposon Control by tRNA-Derived Small RNAs. Cell 2017, 170(1):61-71 e11.[PMID: 28666125]

  15. Maute RL et al: tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci U S A 2013, 110(4):1404-1409.[PMID: 23297232]

  16. Ruggero K et al: Small noncoding RNAs in cells transformed by human T-cell leukemia virus type 1: a role for a tRNA fragment as a primer for reverse transcriptase. J Virol 2014, 88(7):3612-3622.[PMID: 24403582]

  17. Falconi M et al: A novel 3'-tRNA(Glu)-derived fragment acts as a tumor-suppressor in breast cancer by targeting nucleolin. FASEB J 2019:fj201900382RR.[PMID: 31560576]

  18. Zhou K et al: A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells. Oncotarget 2017, 8(56):95377-95391.[PMID: 29221134]

  19. Goodarzi H et al: Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015, 161(4):790-802.[PMID: 25957686]

  20. Natt D et al: Human sperm displays rapid responses to diet. PLoS Biol 2019, 17(12):e3000559.[PMID: 31877125]

  21. Veneziano D et al: Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2019, 116(48):24252-24258.[PMID: 31723042]

  22. Haussecker D et al: Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 2010, 16(4):673-695.[PMID: 20181738]

  23. Balatti V et al: tsRNA signatures in cancer. Proc Natl Acad Sci U S A 2017, 114(30):8071-8076.[PMID: 28696308]

  24. Cho H et al: Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3' trailer-derived tRNA fragments. Nucleic Acids Res 2019, 47(18):9888-9901.[PMID: 31504775]

  25. Babiarz JE et al: Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev 2008, 22(20):2773-2785.[PMID: 18923076]

  26. Hasler D et al: The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway. Mol Cell 2016, 63(1):110-124.[PMID: 27345152]

  27. Pekarsky Y et al: Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer. Proc Natl Acad Sci U S A 2016, 113(18):5071-5076.[PMID: 27071132]

  28. Liao JY et al: Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3' trailers. PLoS One 2010, 5(5):e10563.[PMID: 20498841]

  29. La Ferlita A et al: Identification of tRNA-derived ncRNAs in TCGA and NCI-60 panel cell lines and development of the public database tRFexplorer. Database (Oxford) 2019, 2019.[PMID: 31735953]

  30. Honda S et al: Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci U S A 2015, 112(29):E3816-3825.[PMID: 26124144]

  31. Donovan J, Rath S, Kolet-Mandrikov D, Korennykh A: Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery. RNA 2017, 23(11):1660-1671.[PMID: 28808124]

  32. Hanada T et al: CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 2013, 495(7442):474-480.[PMID: 23474986]

  33. Saikia M et al: Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress. Mol Cell Biol 2014, 34(13):2450-2463.[PMID: 24752898]

  34. Wang Q et al: Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Mol Ther 2013, 21(2):368-379.[PMID: 23183536]

  35. Deng J et al: Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism. Mol Ther 2015, 23(10):1622-1629.[PMID: 26156244]

  36. Zhou J et al: Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection. J Gen Virol 2017, 98(7):1600-1610.[PMID: 28708049]

  37. Yang X et al: 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 2017, 27(5):606-625.[PMID: 28418038]

  38. Ivanov P et al: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011, 43(4):613-623.[PMID: 21855800]

  39. Ivanov P et al: G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments. Proc Natl Acad Sci U S A 2014, 111(51):18201-18206.[PMID: 25404306]

  40. Schaffer AE et al: CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 2014, 157(3):651-663.[PMID: 24766810]

  
  

• 數據分析包括可直接使用的關鍵數據、丰富的注释信息和出版级别的图形。

  差異修飾small RNA列表，包括miRNA，pre-miRNA和tsRNA（tRF＆tiRNA）

  
  

  MatureID:  成熟miRNA在miRBase 的ID

  Group m7G miRNA level (normalized, log2): 基於Cy5标记的m7G-IP RNA的初始信号值得到的log2转换的标准化后的组平均值。

  Treated, Control:  实验组和对照组

  FC:  兩組比較的差異倍數

  P:  t test检验分析的统计学差异的p值

  Regulation: 兩組比較的上調或者下調

  Group m7G %modified miRNA: m7G修饰的miRNA组平均百分比

  miRNA_family: 具有相同的種子序列的miRNA家族

  m7G_motif: “RAm7GGT” m7G 的motif基序, R 代表G或A

  
  

  差異修飾的miRNA，pre-miRNA和tsRNA（tRF＆tiRNA）的分层聚类热图

  
  

  
  

  图1.差异修饰small RNA的分层聚类热图。 修饰的RNA水平由左上方小图中红蓝色色标表示。顶部树状图显示了样品之间修饰图谱相对接近度。组别由热图上方的色条表示。