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Aug 2020

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U2.3 Precursor Small Nuclear RNA in vitro Processing Assay
U2.3前体小核 RNA体外加工试验   

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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

  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

  1. Assemble pre-U2.3 templates

    1. Add the following to the PCR reaction:

      36.5 μl ddH2O

      10 μl 5× Phusion HF buffer

      1 μl 10 mM dNTP mix

      200 ng DNA of Arabidopsis

      0.5 μl U2.3-RNA-F and U2.3-RNA-R primers (10 pmol/μl)

      0.5 μl High-Fidelity DNA Polymerase

    2. Perform PCR to generate pre-U2.3 fragment with the following cycles:

      98°C for 30 s to denature the DNA

      95°C for 10 s

      58°C for 30 s

      72°C for 15 s

      Repetition of PCR for 31 cycles

      72°C for 10 min

      4°C for storage

    3. Electrophorese PCR-generated DNA fragments in 1.5% agarose gel.

    4. Verify the pre-U2.3 (355 bp) fragment and purify PCR product from agarose gel using Gel Purification kit following manufacturer’s instructions.

    5. Cloning of the amplified PCR products to pCR-Blunt.

      6 μl purified pre-U2.3 PCR product

      1 μl pCR-Blunt vector

      2 μl 5× T4 DNA Ligase buffer

      1 μl T4 DNA Ligase

    6. Incubate the ligation reaction at room temperature for 10 min. Transform the construct into competent Escherichia coli. Extract the positive vector and confirm the pre-U2.3 fragment using singer sequencing.

    7. Assemble pre-U2.3 templates and fuse with T7 promoter and Ploy-G. Add the following to the reaction:

      74 μl ddH2O

      20 μl 5× Phusion HF buffer

      2 μl 10 mM dNTP mix

      1 μl pre-U2.3 pCR-Blunt vector (50 ng/μl)

      1 μl T7-U2.3-F and PolyG-U2.3-R primers (10 pmol/μl)

      1 μl High-Fidelity DNA Polymerase

      Perform PCR with the following cycles:

      98°C for 30 s to denature the DNA

      95°C for 10 s

      58°C for 30 s

      72°C for 15 s

      Repetition of PCR for 33 cycles

      72°C for 10 min

      4°C for storage

    8. Load a small aliquot of PCR product (5 μl) in 1.5% agarose gel for checking the quality of PCR fragments. If the product contains a single band, the rest of the product (95 μl) can be purified directly using the PCR Extraction Kit without gel purification.

    9. Elute the PCR product in the final 50 μl DEPC water, used as in vitro transcription template.


  2. Synthesize pre-U2.3-polyG snRNA substrates

    1. In vitro RNA transcription. Add the following to the reaction:

      22 μl DEPC water

      10 μl 5× Transcription Buffer (Ambion)

      2.5 μl NTPs (10 mM ATP, CTP, UTP, GTP)

      1 μl RNasin (Promega 40 units/μl)

      1 μl 1 M DTT

      5 μl Transcription Template (200 ng/μl)

      1 μl T7 RNA Polymerase (50 unit/μl)

      Incubate for 1 h at 37°C

    2. Add 1 μl of DNase I (10 units/μl) and incubate for 15 min at 37°C.

    3. Add 50 μl 2× formamide loading buffer to the reaction solution, boil for 5 min, and load onto a pre-run 5% denaturing polyacrylamide gel containing 6 M urea. Separate RNAs at 20 mA/gel, ~1 h.

    4. After electrophoresis, visualize the PAGE gel with ethidium bromide and cut out the RNA bands directly with a clean razor blade. The gel slice is crushed into a fine slurry, which is soaked in the TE buffer. The slurry is then centrifuged and the supernatant recovered with a pipette. Polyacrylamide fragments carried over into the supernatant can be removed by filtration through a 0.2 mm filter.

    5. Add 3 M sodium acetate (pH 5.2) to a final concentration of 0.3 M and use 3 volumes of absolute ethanol to precipitate RNA. Pellet the RNA by centrifuging at 12,000 × g for 15 min.

    6. Wash small pellets with 75% ethanol to remove undesired salts.

    7. Dissolve the RNA substrates in 20 μl DEPC water.

    8. Radioactive labeling of RNA substrates. Add the following to the reaction:

      3 μl DEPC H2O

      20 μl RNA substrate

      3 µl T4 PNK Reaction Buffer (10×)

      1.5 µl T4 PNK

      1.5 μl RNasin (Promega 40 units/µl)

      1 µl [γ-32P]-ATP

      Incubate at 37°C for 30 min. Add 30 μl DEPC water (This volume can be varied if more or less pre-snRNA probe is desired).

    9. Unincorporated nucleotides are removed using a Sephadex G-25 quick spin column.


  3. Synthesize radioactive RNA marker

    1. Take 15 μl commercial RNA marker into Sephadex G-25 column to remove the salt and dye.

    2. For radioactive labeling of the RNA marker, add the following to the reaction:

      10 μl filtered RNA marker

      6 μl DEPC water

      2 µl T4 PNK Reaction Buffer (10×)

      1 µl T4 PNK

      1 µl [γ-32P]-ATP

      Incubate at 37°C for 30 min

    3. Remove unincorporated nucleotides using a Sephadex G-25 quick spin column.

    4. Prepare a dilution series of the labeled RNA marker (1:5, 1:15, 1:50, and 1:150). Drip 2 µl of series diluted marker and pre-U2.3-polyG RNA substrate onto the nylon membrane. Fix the RNAs spot to the membrane by cross linking using the UV crosslinker with a setting of 120,000 µJ cm-2.

    5. Place the plastic-wrapped nylon membrane on the storage phosphor imaging screen and expose for 8 h. The phosphor screen is then scanned with PhosphorImager.

    6. Dilute RNA marker using RNA loading buffer to same radiation level with pre-U2.3-polyG RNA substrate according to the radioactive signal of the series dilution spot.


  4. In vitro processing assay of pre-U2.3-polyG snRNA

    1. Carefully grind 2 g inflorescence to fine powder in liquid N2 and pour the powder into 15 ml tube.

    2. Set the tube on ice, add 8 ml M1 buffer, and invert several times.

    3. Filter the solution through cell strainer (100 µM) into a 50 ml tube.

    4. Centrifuge at 12,000 × g for 10 min at 4°C.

    5. Discard the supernatant, add 4 ml M2 buffer, and mix by pipetting. Transfer the solution into a 5 ml tube. Wash with 3 ml M2 buffer and 3 ml M3 buffer, consecutively, each wash by spin 1 min at 13,200 × g. Remove all of the solution with a small tip pipette.

    6. Add 200 μl processing buffer (without creatine phosphate) to suspend the pellet.

    7. Take 10 μl nuclear protein into 190 μl Bradford reagent, let stand for 5 min, and measure the protein concentrations at 595 nm.

    8. Dilute the nuclear protein of each sample to 50 ng/μl using processing buffer (without creatine phosphate).

    9. For a total of 50 μl reaction volume for each repeat, incubate 40 μl of nuclear protein, 5 μl of labeled DNA, and 5 μl of 200 mM creatine phosphate stock solution (20 mM final concentration) at 30°C. (The number of repeats can be varied. We did four repeats here).

    10. Stop reaction by adding 450 μl TRIzol at various time points (we stopped at 10, 30, 60, and 90 min reaction time points). RNAs were extracted following the manufacturer’s instructions.

    11. Dissolve the RNA pellet in 10 μl DEPC water. Add 10 μl formamide loading buffer to the solution and boil for 5 min.

    12. Load half of the RNA extraction, as well as 0.5 μl pre-U2.3 substrate (used as input) and 5 μl RNA marker, onto a pre-run 6% denaturing polyacrylamide gel containing 6 M urea.

    13. Run at 120 V in 1× TBE until the loading dye reaches 2/3 of the gel length.

    14. Remove the gel from the electrophoresis apparatus after electrophoresis. Transfer gel onto plastic wrap carefully. Absorb solution with filter paper. Wrap the gel carefully using plastic wrap.

    15. Place the plastic-wrapped gel on the storage phosphor imaging screen and expose for 6-10 h. The phosphor screen is then scanned with the PhosphorImager.

    16. Export the image of the radioactive signal in the ‘tiff’ format (Figure 1).



      Figure 1. In vitro processing of pre-U2.3-polyG. In vitro transcribed pre-U2.3-polyG RNAs were processed in the nuclear protein extracts from Arabidopsis for various time points, as indicated at the top of the figure. After extraction, RNAs were resolved on a PAGE gel and detected with a PhosphorImager. The position of the pre-U2.3-polyG RNA is indicated by the grey arrow, while the processed mature snRNAs are indicated by the black arrow.

Data analysis

Band intensities were quantified with ImageJ or Quantity One software following these steps (we used Quantity One):

  1. Open the gel image using Quantity One.

  2. Open the ‘Volume Tools’ and select the ‘Volume Rect Tool.’ Select minimum areas of target bands from the image, including the processed mature snRNA and input (Figure 2).



    Figure 2. The selected frame of target bands in Quantity One software. Successively select the right processed snRNA bands of various reactions (frame 1-4), according to the relevant position of RNA markers and the pre-U2.3-polyG input (frame 5), using the ‘Volume Rect Tool.’


  3. Click the ‘Volume Analysis Report’ and select report options including area name, concentration, mean value, and density (Table 1).


    Table 1. Intensity of relevant bands

  4. Index Band
    Name
    Volume
    INT*mm2
    Mean Value
    INT
    Density
    INT/mm2
    1 10 min 307.75 33.13 4621.05
    2 30 min 476.26 58.59 8173.07
    3 60 min 694.97 73.95 10315.99
    4 90 min 1095.37 116.56 16259.60
    5 Input 829.91 72.58 10125.62

  5. Use the value of Density to quantify the relative processing efficiency. The intensities of the radioactive signals of various mature U2.3 were normalized to the input.

Recipes

  1. Processing buffer

    10 mM HEPES, pH 7.9

    50 mM KCl

    10% glycerol

    20 mM creatine phosphate

    3 mM MnCl2

    2.5% Polyvinyl alcohol

    1 mM DTT

    Add PMSF, Pepstatin A, and protease inhibitor just before use.

    Note: Be sure to make the solution on the day of use.

  2. M1 buffer

    10 mM Phosphate buffer, pH 7.8

    0.1 M NaCl

    10 mM 2-Mercaptoethanol

    1 M Hexylene glycol

    Add PMSF, Pepstatin A, and protease inhibitor just before use.

  3. M2 buffer

    10 mM Phosphate buffer, pH 7.8

    0.1 M NaCl

    10 mM 2-mercaptoethanol

    1 M Hexylene glycol

    10 mM MgCl2

    Add PMSF, Pepstatin A, and protease inhibitor just before use

  4. M3 buffer

    10 mM Phosphate buffer, pH 7.8

    0.1 M NaCl

    10 mM 2-Mercaptoethanol

    Add PMSF, Pepstatin A, and protease inhibitor just before use

  5. 2× formamide loading buffer

    95% Deionized formamide

    0.025% (w/v) Bromophenol blue

    0.025% (w/v) Xylene cyanol FF

    5 mM EDTA, pH 8.0

  6. 6% denaturing polyacrylamide gel containing 6 M urea (10 ml)

    2 ml 30% polyacrylamide solution (29:1)

    Urea 3.6 g

    1ml 5× TBE

    9.9 ml water

    80 µl 10% APS

    10 µl TEMED

    Before preparing the gel, clean all components with 70% ethanol and RNase away.

Acknowledgments

This work was supported by grants from the State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources (SKLCUSA-a202008).

Competing interests

The authors declare no competing interests.

References

  1. Baillat, D., Hakimi, M. A., Naar, A. M., Shilatifard, A., Cooch, N. and Shiekhattar, R. (2005). Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123(2): 265-276.
  2. Carbon, P., Murgo, S., Ebel, J. P., Krol, A., Tebb, G. and Mattaj, L. W. (1987). A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell 51(1): 71.
  3. Chen, J. and Wagner, E. J. (2010). snRNA 3' end formation: the dawn of the Integrator complex. Biochem Soc Trans 38(4): 1082-1087.
  4. Hernandez, N. (2001). Small nuclear RNA genes: a model system to study fundamental mechanisms of transcription. J Biol Chem 276(29): 26733-26736.
  5. Jawdekar, G. W. and Henry, R. W. (2008). Transcriptional regulation of human small nuclear RNA genes. Biochimica et Biophysica Acta 1779(5): 295-305.
  6. Liu, Y., Li, S., Chen, Y., Kimberlin, A. N., Cahoon, E. B. and Yu, B. (2016). snRNA 3’ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis. PLoS Biology 14(10): e1002571.
  7. Vankan, P., Filipowicz, W. (1988). Structure of U2 snRNA genes of Arabidopsis thaliana and their expression in electroporated plant protoplasts. EMBO J 7(3): 791.
  8. Uguen, P., Murphy, S. (2003). The 3' ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing. EMBO J 22(17): 4544-4554.
  9. Uguen, P., Murphy, S. (2004). 3'-box-dependent processing of human pre-U1 snRNA requires a combination of RNA and protein co-factors. Nucleic Acids Res 32(10): 2987-2994.

简介

[摘要]小核 RNA (snRNAs)对真核细胞活动至关重要,并在前体 mRNA 剪接中发挥重要作用。上的分子机制吨他snRNA启动的转录,通过上行/下行的顺式元件和反式相关元素调节,已经研究详细使用无细胞提取物。然而,该处理的前体的snRNA(预snRNA的),其由3'预snRNA启动的端成熟需要,仍然不清楚一个适当的加工法是难以开发体外。在这里,我们提出了一个体外 使用合成标记 RNA 作为底物来研究 pre-snRNA 的 3' 切割的方法。


[背景]作为关键部件小号剪接的用于处理和剪接前体米RNA小号(前mRNA小号),小号商场核RNA(snRNAs )是在真核细胞必需和基本非编码小RNA 。的转录复合物的rel颖的DNA依赖性RNA聚合酶II(POL II)我š需要最snRNA的转录(包括U1,U2,U4,和U5),除了U6 ,其依赖于RNA聚合酶III(POL III) (Carbon et al. , 1987 ; Vankan and Filipowicz, 1988)。Pol II 介导的 snRNA 转录需要后生动物中的一组整合因子 (INT)(Baillat等,2005)。在INT含有至少14个在后生动物亚基(Baillat等人,2005; Chen和瓦格纳,2010)聚合酶II,具有C末端结构域(CTD)相关联,以形成所述转录复合物。最近,涉及snRNA的生物合成5个植物INT因素被确定拟南芥和snRNA的处理(DSP)称为有缺陷的,包括DSP1 - 4和CPSF73-I(切割和聚腺苷酸化特异性因子73 kDa)的(刘等人,2016)。钍ë INT小号/ DSP小号被募集到snRNA启动的启动子和准用聚合酶II以合成预snRNA的,transcrib荷兰国际集团它超过3'成熟snRNAs的端。在新生的预snRNA启动转录包含的snRNA的序列和过量的3 '盒RNA片段。因此,预snRNAs是受3 “成熟,涉及在切割位点的新生转录物的核酸内切裂解的处理位于上游的3 ”盒,以产生成熟的snRNA (Uguen和Murphy,2003和2004; Baillat等al. , 2005; Chen 和 Wagner, 2010)。

的分子机制的根本的snRNA的起始和抑制转录已经很好地研究都在体内和体外。该功能的的顺式元件小号,包括所述远端序列元件(DSE ),近端序列元件(PSE人类)小号和上游序列元件(USE植物)小号,并且所述反式元素,包括INT小号/ DSP小号, 进行了详细的调查(Hernandez , 2001)。

COMPAR编带snRNA启动转录的充分研究调节,预snRNA启动的处理步骤仍然知之甚少(Hernandez的,2001年; Jawdekar和Henry ,2008) 。大多数关于pre-snRNA加工的研究是通过评估特定基因型材料中 pre-snRNA 的相对含量来进行的。一个有效的系统用于使用细胞提取物将有利于检测预snRNA启动的处理的研究的预snRNA启动的在成熟特定基因型的植物和揭示的影响特定因素对预RNA成熟转录后。

因此,我们应用一个合成的预U2.3 SN RNA和创建的系统来分析在体外使用预U2.3的处理活动的生产细胞提取物小号。个是我Ñ v ITRO p rocessing分析provid上课检测的活化的方法预snRNA的成熟。

关键字:核小RNA, Pre-snRNA, 加工, 拟南芥, 活体外

 
材料和试剂
 
1.尼龙膜(Invitrogen,目录号:LC2003)      
2. DNA酶I (NEB ,目录号:M0303S)      
3.无核酸酶W¯¯亚特(赛默飞世科学,目录号:AM9932)      
4. Trizol(ThermoFisher Scientific,目录号:10296028)      
5.氯仿(Sigma-Aldrich,目录号:288306 )      
6.异丙醇(Sigma-Aldrich,目录号:563935 )      
7.乙醇(Sigma-Aldrich,目录号:51976 )      
8. 2-米ercaptoethanol (Gibco公司,目录号:21985023 )      
9.己克lycol(Sigma-Aldrich公司,目录麻木ER:112100)      
10. Triton X-100(Fisher Scientific,Acros Organics,目录号:215680010)   
11. dNTP (各10 mM)(ThermoFisher Scientific,目录号:R0192 )   
12. NTP (每个 10 mM)(ThermoFisher Scientific,分子生物学级 NTP ,目录号:R0481 )   
13. G lycerol(Sigma-Aldrich,目录号:G5516 )   
14. HEPES(Sigma-Aldrich,目录号:H3375)   
15.各种盐:氯化钠,氯化钾,氯化镁2 ,和的MnCl 2 (Sigma-Aldrich公司,目录号小号:S9888 ,P9541 ,M8266 ,和M1787分别)   
16. Ç reatine磷酸盐(Sigma-Aldrich公司,目录号:10621714001)   
17.聚乙烯醇(Sigma-Aldrich,目录号:341584 )   
18. DTT (ThermoFisher Scientific,目录号:R0861 )   
19.胃酶抑素A(Sigma-Aldrich,目录号:P5318 )   
20.苯基甲基磺酰基(PMSF)(Sigma-Aldrich,目录号:52332 )   
21.蛋白酶抑制剂(Roche,目录号:11697498001)   
22.二乙基p yrocarbonate(DEPC)(Sigma-Aldrich公司,目录号:D5758)   
23. [ γ - 32 P]-ATP(3,000 Ci/mmol,10 mCi/ml)(PerkinElmer,目录号:BLU002A)   
24. T7 RNA p olymerase (赛默飞,目录号:18033019)   
25. T4 p olynucleotide ķ inase (PNK)(NEB,目录号:M0201S)   
26.高˚F idelity DNA p olymerase (赛默飞,目录号:F530L)   
27. p CR-Blunt (ThermoFisher,目录号:K270020)   
一个PCR-钝的地图可以发现在上的网站的制造商:www.thermofisher.com/order/catalog/product/K270020#/K270020。
28. RNA米arker (Abnova公司,目录号:R0002)   
29.布拉德福德ř eagent (生物太阳神d,目录号:5000205)   
30.凝胶纯化试剂盒   
31. PCR 提取试剂盒   
32. RNase   
33.引物:   
U 2.3-RNA-F : atacctttctcggccttttggc
U 2.3-RNA-R : ctgcgtaacatatataaatatctctg             
T7-U2.3-F:TAATACGACTCACTATAGGGatacctttctcggc
PolyG-U2.3-R: CCCCCCCCCCCC ctgcgtaacatatataa             
34.处理缓冲器(见ř ecipes)   
35. M1缓冲器(见ř ecipes)   
36. M2缓冲器(见ř ecipes)   
37. M3缓冲器(见ř ecipes)   
38. 2 ×甲酰胺上样缓冲液(见ř ecipes)   
39.含有 6 M 尿素的 6% 变性聚丙烯酰胺凝胶(见配方)   
 
设备
 
PhosphorImager (GE Health Care,型号:Typhoon FLA 9500)
适合处理32 P 放射性的空间
盖革计数器 (Thermo, 900 mini)
保护用户免受放射性侵害的有机玻璃防护罩
用于32 P 废物的有机玻璃盒
Sephadex G-25 离心柱(默克公司,11273990001)
聚合酶链反应机
荧光屏(分子动力学)
细胞过滤器(Biofil,目录号:CSS013100)
垂直电泳凝胶盒和带有垫片和梳子的玻璃板(Bio- Ra d,型号:Mini-PROTEAN Tetra)
分光光度计(一个NY UV吸收分光光度法包括布拉德福德可用)
 
软件
 
数量一(Bio-Rad 实验室)或I mageJ
 
程序
 
组装U2.3 之前的模板
将以下内容添加到 PCR 反应中:
36.5 μ升的DDH 2 ö
10 μ l 5 × Phusion HF 缓冲液
1 μ l 10 mM dNTP 混合物
200 ng拟南芥DNA
0.5 μ l U 2.3-RNA-F和U 2.3-RNA-R引物(10 pmol/ μ l)
0.5 μ升高保真DNA聚合酶
执行 PCR 以生成具有以下循环的 pre-U2.3 片段:
98 °C 30 秒使 DNA 变性
95 °C 10 秒
58 °C 30 秒
72 °C 15 秒
重复 PCR 31 个循环
72 °C 10 分钟
4 °C储存
在 1.5% 琼脂糖凝胶中电泳 PCR 产生的 DNA 片段。
按照制造商的说明,使用凝胶纯化试剂盒验证前 U2.3(355 bp)片段并从琼脂糖凝胶中纯化 PCR 产物。
将扩增的 PCR 产物克隆到 pCR-Blunt。
6 μ升纯化预U2.3 PCR产物
1 μ l pCR-Blunt 载体
2 μ l 5 × T4 DNA 连接酶缓冲液
1 μ升T4 DNA连接酶
在室温下孵育连接反应 10 分钟。变换构建成主管ê scherichia大肠杆菌。提取阳性载体并使用歌手测序确认 pre-U2.3 片段。
组装U2.3 之前的模板并与 T7 启动子和 Ploy-G 融合。将以下内容添加到反应中:
74 μ升的DDH 2 ö
20 μ l 5 × Phusion HF 缓冲液
2 μ l 10 mM dNTP 混合物
1 μ l pre-U2.3 pCR-Blunt 载体(50 ng/ μ l)
1 μ l T7-U2.3-F 和PolyG-U2.3-R 引物(10 pmol/ μ l)
1 μ升高保真DNA聚合酶
使用以下循环进行 PCR:
98 °C 30 秒使 DNA 变性
95 °C 10 秒
58 °C 30 秒
72 °C 15 秒
重复 PCR 33 个循环
72 °C 10 分钟
4 °C储存
加载PCR产物(5小等分μ在1.5%升)琼脂糖凝胶用于检查的PCR片段的质量。如果产品包含一个单一条带,其余的的产物(95 μ升),可直接使用纯化的PCR提取试剂盒凝胶未经纯化。
洗脱在最后50中的PCR产物μ升DEPC水,用作体外转录的模板。
 
合成 pre-U2.3-polyG snRNA 底物
体外RNA 转录。将以下内容添加到反应中:
22 μl DEPC 水
10 μl 5 ×转录缓冲液(Ambion)
2.5 μl NTP(10 mM ATP、CTP、UTP、GTP)
1微升RNA酶抑制剂(Promega公司40个单位/ μ升)
1 微升 1 M DTT
5 μl 转录模板 (200 ng/μl)
1 μl T7 RNA 聚合酶(50 单位/μl)
在 37°C 下孵育 1小时
加入 1 μl DNase I(10 单位/μl)并在 37°C 下孵育 15 分钟。
加入50μl2 ×甲酰胺上样缓冲液到反应溶液中,煮沸5分钟,并加载到预运行5%变性聚丙烯酰胺凝胶含有荷兰国际集团6M尿素。以 20 mA/凝胶分离 RNA,约 1 小时。
电泳后,用溴化乙锭对 PAGE 凝胶进行可视化,并用干净的刀片直接切出 RNA 带。凝胶切片被粉碎成细浆,浸泡在 TE 缓冲液中。然后离心浆液并用移液管回收上清液。携带到上清液中的聚丙烯酰胺片段可以通过 0.2 毫米过滤器过滤去除。
添加 3 M 醋酸钠 (pH 5.2) 至最终浓度为 0.3 M,并使用 3 倍体积的无水乙醇沉淀 RNA。以 12 , 000 × g离心15 分钟,使 RNA 沉淀。
洗小粒料小号用75%乙醇,以除去不需要的盐。
溶解在20所述RNA底μ升DEPC水。
无线电RNA底物的有源标签。将以下内容添加到反应中:
3 微升 DEPC H 2 O
20 μl RNA 底物
3 µl T4 PNK 反应缓冲液 (10 × )
1.5 微升 T4 PNK
1.5 μl RNasin(Promega 40 单位/μl)
1 µl [ γ - 32 P]-ATP
在 37°C 下孵育 30 分钟。添加 30 μl DEPC 水(如果需要更多或更少的 pre-snRNA 探针,可以改变此体积)。
未掺入的核苷酸被使用除去一个葡聚糖G-25离心柱。
 
合成放射性 RNA 标记
取15 μ升商业RNA标记插入的Sephadex G-25柱,以除去盐和染料。
当r的RNA标记的adioactive标记,一个DD以下反应:
10 μ升筛选RNA标记
6 μ升DEPC水
2 µl T4 PNK 反应缓冲液 (10 × )
1 微升 T4 PNK
1 µl [ γ - 32 P]- ATP
在 37 °C 下孵育30 分钟
拔下U nincorporated使用核苷酸一葡聚糖G-25离心柱。
制备系列稀释的该标记的RNA标记(1:5,1:15,1:50,和1:150)。将 2 µl系列稀释标记物和 pre-U2.3-polyG RNA 底物滴到尼龙膜上。使用通过交联固定的RNA点到膜的UV交联剂与一个120设置,000 μJ厘米- 2 。
放置在塑料包装的尼龙膜的存储荧光体摄像画面和暴露8小时。然后用 PhosphorImager 扫描荧光屏。
使用RNA加样缓冲液以相同的辐射电平与预U2.3-多聚G RNA底物稀RNA标记根据对所述的放射性信号系列稀释斑点。
 
pre-U2.3-polyG snRNA 的体外加工测定
小心地将 2 g 花序在液氮2 中研磨成细粉,然后将粉末倒入 15 ml 管中。
将试管置于冰上,加入 8 ml M1 缓冲液,并颠倒数次。
通过细胞过滤器 (100 µ M )将溶液过滤到 50 ml 管中。
在 4°C 下以 12 , 000 × g离心10 分钟。
弃去上清液,加入 4 ml M2 缓冲液,移液混合。将溶液转移到 5 ml 管中。用 3 ml M2 缓冲液和 3 ml M3 缓冲液连续洗涤,每次以 13 度、200 × g旋转 1 分钟洗涤。用小吸头移液器除去所有溶液。
添加 200 μl 处理缓冲液(不含磷酸肌酸)以悬浮沉淀。
采取10微升的核蛋白入190微升Bradford试剂,静置为5分钟,并且在595nm处测量蛋白浓度。
使用处理缓冲液(不含磷酸肌酸)将每个样品的核蛋白稀释至 50 ng/μl。
对于总的50微升反应体积为每个重复,孵化40微升的核内蛋白,5微升的标记的DNA,和5μl的在30℃的200mM磷酸肌酸股票溶液(20mM终浓度)。(该号码的重复可以改变的。我们做了四个重复这里)。
通过在不同时间点加入450μl的TRIzol试剂停止反应小号(瓦特Ë停止在10,30,60,和90个分钟反应时间点)。按照制造商的说明提取 RNA 。
溶解于10 RNA沉淀μ升DEPC水。添加10 μ升甲酰胺上样缓冲液的溶液并煮沸5分钟。
负荷一半的RNA提取的,以及0.5 μ升预U2.3底物(用作输入)和5 μ升RNA标记物,在6%变性聚丙烯酰胺凝胶含有6M尿素的预运行。
在 1 × TBE 中以 120 V 运行,直到加载染料达到凝胶长度的2/3 。
电泳后从电泳仪中取出凝胶。小心地将凝胶转移到塑料包装上。用滤纸吸收溶液。用保鲜膜小心地包裹凝胶。
放置在塑料包装的凝胶的存储荧光体摄像画面和暴露6 - 10小时。荧光屏然后用扫描的磷光。
导出的图像的ř adioactive信号在“TIFF”格式(图1)。
 
 
图 1. pre-U2.3-polyG 的体外加工。体外转录的预U2.3-多聚G的RNA在核蛋白质提取物处理来自拟南芥的各种时间点小号,所指示的在顶部的图中。萃取后,将RNA上解决一PAGE凝胶,并用检测到的一个磷光。预U2.3-多聚G RNA的位置由表示的灰色箭头,而加工的成熟snRNA的小号是指示由所述黑色箭头。
 
数据分析
 
乙和强度分别为q uantifi版用ImageJ的或量的一种软件后续荷兰国际集团这些步骤(我们使用量的一种):
使用数量一打开凝胶图像。
打开“音量工具”并选择“音量矩形工具” 。从图像中选择目标带的最小区域,包括处理后的成熟 snRNA 和输入(图 2)。
 
 
图2.该目标频带的数量在One软件选择的帧。依次选择的各种反应的权利处理snRNA的频带小号(帧1 - 4) ,根据对RNA标记物的相关位置和预U2.3-多聚G输入(框5) ,使用“音量矩形工具”。
 
Ç舔“量分析报告” ,并选择报告选项,包括区域名称,浓度,均值,和密度(表1) 。
 
表1.我相关频段的ntensity
 
ü自身的值密度到孔定量˚F y中的相对处理效率。所述intensit IES的所述放射性信号š的各种成熟U2.3被归一化到的输入。
 
食谱
 
处理缓冲区
10 mM HEPES ,pH 7.9
50 毫米氯化钾
10% 甘油
20 mM 磷酸肌酸
3 毫米氯化锰2
2.5% 聚乙烯醇
1 毫米 DTT
加入PMSF,胃蛋白酶抑制剂A ,而只是摆在我们面前的蛋白酶抑制剂ê 。
注意:请务必在使用当天制作溶液。
M1缓冲器
10 mM磷酸盐缓冲液,pH 7. 8
0.1中号的Na氯
10 mM 2-巯基乙醇
1 M己二醇
加入PMSF,胃蛋白酶抑制剂A ,而只是摆在我们面前的蛋白酶抑制剂ê 。
M2缓冲器
10 mM磷酸盐缓冲液,pH 7. 8
0.1中号的Na氯
10毫2-米ercaptoethanol
1 M己二醇
10 毫米氯化镁2
加入PMSF,胃蛋白酶抑制剂A ,而只是摆在我们面前的蛋白酶抑制剂Ë
M3缓冲器
10 mM磷酸盐缓冲液,pH 7. 8
0.1中号的Na氯
10 mM 2-巯基乙醇
加入PMSF,胃蛋白酶抑制剂A ,而只是摆在我们面前的蛋白酶抑制剂Ë
2 ×甲酰胺上样缓冲液
95% 去离子甲酰胺             
0.025% (w/v) 溴酚蓝             
0.025% (w/v) 二甲苯氰 FF             
5 mM EDTA,pH 8.0             
含有 6 M 尿素的 6% 变性聚丙烯酰胺凝胶(10 毫升)
2 毫升30% 聚丙烯酰胺溶液 (29:1)             
尿素 3.6 克
1ml 5 × TBE
9.9 毫升水
80 微升 10% APS
10 微升 TEMED
在制备凝胶之前,用 70% 乙醇和 RNase 清洁所有组件。
 
致谢
 
这项工作是支持摹从国家重点实验室保护的咆哮和亚热带农业生物资源(SKLCUSA-a202008)的利用。
 
利益争夺
 
作者声明没有竞争利益。
 
参考
 
Baillat, D. , Hakimi, MA , Naar, AM , Shilatifard, A. , Cooch, N.和Shiekhattar, R. ( 2005 ) 。Integrator 是一种小核 RNA 加工的多蛋白介质,与 RNA 聚合酶 II 的 C 端重复序列相关联。单元格123(2):265-276。
碳,P 。,穆尔古木尔格,S 。,玉宝,JP ,克罗尔,A 。,Tebb,G 。和Mattaj, LW ( 1987 ) 。常见的八聚体基序结合蛋白参与 RNA 聚合酶 III 对 U6 snRNA 的转录和 RNA 聚合酶 II 对 U2 snRNA 的转录。单元格51(1):71。
Chen, J.和Wagner, EJ ( 2010 ) 。snRNA 3' 末端形成:Integrator 复合体的曙光。Biochem Soc Trans 38(4):1082-1087。
埃尔南德斯,N. (2001 年)。小核 RNA 基因:研究转录基本机制的模型系统。J Biol Chem 276(29): 26733-26736。
Jawdekar, GW和Henry, RW ( 2008 ) 。人类小核 RNA 基因的转录调控。Biochimica et Biophysica Acta 1779(5): 295-305。
Liu, Y.、Li, S.、Chen, Y.、Kimberlin, AN、Cahoon, EB 和 Yu, B. (2016 年)。拟南芥中含有 CPSF73 的复合物对 snRNA 3' 末端进行加工。PLoS 生物学14(10):e1002571。
Vankan, P., Filipowicz, W. ( 1988 ) 。拟南芥 U2 snRNA 基因的结构及其在电穿孔植物原生质体中的表达。EMBO J 7(3): 791。
Uguen, P., Murphy, S. ( 2003 ) 。人类 pre-snRNA 的 3' 端由 RNA 聚合酶 II CTD 依赖的 RNA 加工产生。EMBO J 22(17):4544-4554。
Uguen, P., Murphy, S. ( 2004 ) 。人类 pre-U1 snRNA 的 3'-box 依赖加工需要 RNA 和蛋白质辅因子的组合。核酸研究32(10):2987-2994。
 
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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.
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