U2.3前体小核 RNA体外加工试验

Chan  Lin; Yujie  Feng; Xueyan  Peng; Jiaming  Wu; Weili  Wang; Yunfeng  Liu

doi:10.21769/BioProtoc.4142

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Peer-reviewed

U2.3 Precursor Small Nuclear RNA in vitro Processing Assay

U2.3前体小核 RNA体外加工试验

WW Weili Wang email

YL Yunfeng Liu email

(*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: 2017

评审: Zhibing LaiSwati MeghaAnonymous reviewer(s)

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

Aug 2020

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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)

Pre-snRNA (Pre-snRNA)

Processing (加工)

Arabidopsis (拟南芥)

In vitro (活体外)

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

Nylon membrane (Invitrogen, catalog number: LC2003)
DNaseI (NEB, catalog number: M0303S)
Nuclease-free water (ThermoFisher Scientific, catalog number: AM9932)
Trizol (ThermoFisher Scientific, catalog number: 10296028)
Chloroform (Sigma-Aldrich, catalog number: 288306)
Isopropanol (Sigma-Aldrich, catalog number: 563935)
Ethanol (Sigma-Aldrich, catalog number: 51976)
2-mercaptoethanol (Gibco, catalog number: 21985023)
Hexylene glycol (Sigma-Aldrich, catalog number: 112100)
Triton X-100 (Fisher Scientific, Acros organics, catalog number: 215680010)
dNTP (10 mM each) (ThermoFisher Scientific, catalog number: R0192)
NTP (10 mM each) (ThermoFisher Scientific, Molecular biology grade NTPs, catalog number: R0481)
Glycerol (Sigma-Aldrich, catalog number: G5516)
HEPES (Sigma-Aldrich, catalog number: H3375)
Various salts: NaCl, KCl, MgCl₂, and MnCl₂ (Sigma-Aldrich, catalog numbers: S9888, P9541, M8266, and M1787, respectively)
Creatine phosphate (Sigma-Aldrich, catalog number: 10621714001)
Polyvinyl alcohol (Sigma-Aldrich, catalog number: 341584)
DTT (ThermoFisher Scientific, catalog number: R0861)
Pepstatin A (Sigma-Aldrich, catalog number: P5318)
Phenylmethylsulfonyl (PMSF) (Sigma-Aldrich, catalog number: 52332)
Protease inhibitor (Roche, catalog number:11697498001)
Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758)
[γ-³²P]-ATP (3,000 Ci/mmol, 10 mCi/ml) (PerkinElmer, catalog number: BLU002A)
T7 RNA polymerase (ThermoFisher, catalog number: 18033019)
T4 polynucleotide kinase (PNK) (NEB, catalog number: M0201S)
High-fidelity DNA polymerase (ThermoFisher, catalog number: F530L)
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.
RNA marker (Abnova, catalog number: R0002)
Bradford reagent (Bio-Rad, catalog number: 5000205)
Gel Purification kit
PCR Extraction Kit
RNase away
Primers:
U2.3-RNA-F: atacctttctcggccttttggc
U2.3-RNA-R: ctgcgtaacatatataaatatctctg
T7-U2.3-F: TAATACGACTCACTATAGGGatacctttctcggc
PolyG-U2.3-R: CCCCCCCCCCCCctgcgtaacatatataa
Processing buffer (see Recipes)
M1 buffer (see Recipes)
M2 buffer (see Recipes)
M3 buffer (see Recipes)
2× formamide loading buffer (see Recipes)
6% denaturing polyacrylamide gel containing 6 M urea (see Recipes)

Equipment

PhosphorImager (GE Health Care, model: Typhoon FLA 9500)
Suitable space for working with ³²P radioactivity
Geiger counter (Thermo, 900 mini)
Plexiglass shield to protect user from radioactivity
Plexiglass box for ³²P waste
Sephadex G-25 spin column (Merck, 11273990001)
PCR Machine
Phosphor Screen (Molecular Dynamics)
Cell strainer (Biofil, catalog number: CSS013100)
Vertical electrophoresis gel box and glass plates with spacers and combs (Bio-Rad, model: Mini-PROTEAN Tetra)
Spectrophotometer (any UV absorption spectrophotometry including Bradford available)

Software

Quantity One (Bio-Rad Laboratories) or ImageJ

Procedure

English

中文翻译

文章信息

版权信息

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.