Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

U2.3 Precursor Small Nuclear RNA in vitro Processing Assay

U2.3前体小核 RNA体外加工试验

CL Chan Lin *
YF Yujie Feng *
XP Xueyan Peng
JW Jiaming Wu
WW Weili Wang
YL Yunfeng Liu

 (*contributed equally to this work) 发布: 2021年09月05日第11卷第17期 DOI: 10.21769/BioProtoc.4142 浏览次数: 2918

评审: Zhibing LaiSwati MeghaAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Aug 2020

Advertisement

“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用来源于最小前体的合成反式siRNA实现植物中高效的基因沉默

利用来源于最小前体的合成反式siRNA实现植物中高效的基因沉默

Adriana E. Cisneros [...] Alberto Carbonell

2025年10月20日 2067 阅读

Turbo-RIP：基于 TurboID 的 RNA 免疫纯化方法，用于绘制植物生物分子凝聚体中的 RNA 图谱

Turbo-RIP：基于 TurboID 的 RNA 免疫纯化方法，用于绘制植物生物分子凝聚体中的 RNA 图谱

Zhi Zhang [...] Panagiotis Nikolaou Moschou

2026年02月05日 852 阅读

DiRT v2.0：用于检测植物双顺反子 tRNA-mRNA 转录本的优化流程

DiRT v2.0：用于检测植物双顺反子 tRNA-mRNA 转录本的优化流程

Fei Zheng [...] Rakesh David

2026年06月20日 244 阅读

Abstract

Small nuclear RNAs (snRNAs) are vital for eukaryotic cell activities and play important roles in pre-mRNA splicing. The molecular mechanism underlying the transcription of snRNA, regulated via upstream/downstream cis-elements and relevant trans-elements, has been investigated in detail using cell-free extracts. However, the processing of precursor snRNA (pre-snRNA), which is required by 3’ end maturation of pre-snRNA, remains unclear as a proper processing assay is difficult to develop in vitro. Here, we present an in vitro method using synthetic labeled RNA as substrates to study the 3’ cleavage of pre-snRNA.

Keywords: snRNA (核小RNA) search Pre-snRNA (Pre-snRNA) search Processing (加工) search Arabidopsis (拟南芥) search In vitro (活体外) search

Background

As critical components of spliceosome for the processing and splicing of precursor mRNAs (pre-mRNAs), small nuclear RNAs (snRNAs) are essential and fundamental non-coding small RNAs in eukaryotic cells. The transcription complex relying on the DNA-dependent RNA polymerase II (Pol II) is required for most snRNA transcription (including U1, U2, U4, and U5), except for U6, which depends on RNA polymerase III (Pol III) (Carbon et al., 1987; Vankan and Filipowicz, 1988). The Pol II mediated transcription of snRNA requires a set of integrator factors (INTs) in metazoans (Baillat et al., 2005). INTs contain at least 14 subunits in metazoans (Baillat et al., 2005; Chen and Wagner, 2010), associated with C-terminal domain (CTD) of Pol II to form the transcription complex. Recently, five plant INT factors involved in snRNA biosynthesis were identified in Arabidopsis and termed Defective in snRNA Processing (DSP), including DSP1-4 and CPSF73-I (Cleavage and Polyadenylation Specificity Factor 73 kDa) (Liu et al., 2016). The INTs/DSPs are recruited to the promoter of snRNA and associate with Pol II to synthesize the pre-snRNA, transcribing it beyond the 3’ end of mature snRNAs. The nascent pre-snRNA transcripts contain the snRNA sequence and the excessive 3’ box RNA fragment. Consequently, pre-snRNAs are subject to 3’ maturation, a process involving endonucleolytic cleavage of the nascent transcript at the cleavage site located on the upstream of the 3’ box, to produce mature snRNA (Uguen and Murphy, 2003 and 2004; Baillat et al., 2005; Chen and Wagner, 2010).


The molecular mechanism underlying the initiation and inhibition of snRNA transcription has been well studied both in vivo and in vitro. The functions of cis-elements, including the distal sequence element (DSE), proximal sequence element (PSE) in humans and upstream sequence element (USE) in plants, and trans-elements, including INTs/DSPs, have been investigated in detail (Hernandez, 2001).


Compared with the well-studied regulation of snRNA transcription, the processing steps of pre-snRNA remain poorly understood (Hernandez, 2001; Jawdekar and Henry, 2008). Most studies on pre-snRNA processing were performed by evaluating the relative content of pre-snRNA in specific genotype material. An efficient system for detecting the processing of pre-snRNA using cell extracts will facilitate the study of the maturation of pre-snRNA in specific genotypic plants and reveal the influence of specific factors on pre-RNA maturation after transcription.


Accordingly, we applied a synthetic pre-U2.3 snRNA and created a system to analyze the in vitro processing activities of pre-U2.3 using the production of cell extracts. This in vitro processing analysis provides a method to detect the activation of pre-snRNA maturity.

Materials and Reagents

  1. Nylon membrane (Invitrogen, catalog number: LC2003)

  2. DNaseI (NEB, catalog number: M0303S)

  3. Nuclease-free water (ThermoFisher Scientific, catalog number: AM9932)

  4. Trizol (ThermoFisher Scientific, catalog number: 10296028)

  5. Chloroform (Sigma-Aldrich, catalog number: 288306)

  6. Isopropanol (Sigma-Aldrich, catalog number: 563935)

  7. Ethanol (Sigma-Aldrich, catalog number: 51976)

  8. 2-mercaptoethanol (Gibco, catalog number: 21985023)

  9. Hexylene glycol (Sigma-Aldrich, catalog number: 112100)

  10. Triton X-100 (Fisher Scientific, Acros organics, catalog number: 215680010)

  11. dNTP (10 mM each) (ThermoFisher Scientific, catalog number: R0192)

  12. NTP (10 mM each) (ThermoFisher Scientific, Molecular biology grade NTPs, catalog number: R0481)

  13. Glycerol (Sigma-Aldrich, catalog number: G5516)

  14. HEPES (Sigma-Aldrich, catalog number: H3375)

  15. Various salts: NaCl, KCl, MgCl2, and MnCl2 (Sigma-Aldrich, catalog numbers: S9888, P9541, M8266, and M1787, respectively)

  16. Creatine phosphate (Sigma-Aldrich, catalog number: 10621714001)

  17. Polyvinyl alcohol (Sigma-Aldrich, catalog number: 341584)

  18. DTT (ThermoFisher Scientific, catalog number: R0861)

  19. Pepstatin A (Sigma-Aldrich, catalog number: P5318)

  20. Phenylmethylsulfonyl (PMSF) (Sigma-Aldrich, catalog number: 52332)

  21. Protease inhibitor (Roche, catalog number:11697498001)

  22. Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758)

  23. [γ-32P]-ATP (3,000 Ci/mmol, 10 mCi/ml) (PerkinElmer, catalog number: BLU002A)

  24. T7 RNA polymerase (ThermoFisher, catalog number: 18033019)

  25. T4 polynucleotide kinase (PNK) (NEB, catalog number: M0201S)

  26. High-fidelity DNA polymerase (ThermoFisher, catalog number: F530L)

  27. pCR-Blunt (ThermoFisher, catalog number: K270020)

    A map of pCR-Blunt can be found on the website of the manufacturer: www.thermofisher.com/order/catalog/product/K270020#/K270020.

  28. RNA marker (Abnova, catalog number: R0002)

  29. Bradford reagent (Bio-Rad, catalog number: 5000205)

  30. Gel Purification kit

  31. PCR Extraction Kit

  32. RNase away

  33. Primers:

    U2.3-RNA-F: atacctttctcggccttttggc

    U2.3-RNA-R: ctgcgtaacatatataaatatctctg

    T7-U2.3-F: TAATACGACTCACTATAGGGatacctttctcggc

    PolyG-U2.3-R: CCCCCCCCCCCCctgcgtaacatatataa

  34. Processing buffer (see Recipes)

  35. M1 buffer (see Recipes)

  36. M2 buffer (see Recipes)

  37. M3 buffer (see Recipes)

  38. 2× formamide loading buffer (see Recipes)

  39. 6% denaturing polyacrylamide gel containing 6 M urea (see Recipes)

Equipment

  1. PhosphorImager (GE Health Care, model: Typhoon FLA 9500)

  2. Suitable space for working with 32P radioactivity

  3. Geiger counter (Thermo, 900 mini)

  4. Plexiglass shield to protect user from radioactivity

  5. Plexiglass box for 32P waste

  6. Sephadex G-25 spin column (Merck, 11273990001)

  7. PCR Machine

  8. Phosphor Screen (Molecular Dynamics)

  9. Cell strainer (Biofil, catalog number: CSS013100)

  10. Vertical electrophoresis gel box and glass plates with spacers and combs (Bio-Rad, model: Mini-PROTEAN Tetra)

  11. Spectrophotometer (any UV absorption spectrophotometry including Bradford available)

Software

  1. Quantity One (Bio-Rad Laboratories) or ImageJ

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lin, C., Feng, Y., Peng, X., Wu, J., Wang, W. and Liu, Y. (2021). U2.3 Precursor Small Nuclear RNA in vitro Processing Assay. Bio-protocol 11(17): e4142. DOI: 10.21769/BioProtoc.4142.

ris ris Download Citation in RIS Format

分类

植物科学 > 植物分子生物学 > RNA

分子生物学 > RNA

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件