RNA聚合酶I活性的体外评估

Marzia  Govoni

doi:10.21769/BioProtoc.2120

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Peer-reviewed

In vitro Assessment of RNA Polymerase I Activity

RNA聚合酶I活性的体外评估

Marzia Govoni email

发布: 2017年02月05日第7卷第3期 DOI: 10.21769/BioProtoc.2120 浏览次数: 8490

评审: HongLok LungGaston A. PizzioAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Feb 2016

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Abstract

In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III without affecting RNA polymerase I (Novello and Stirpe, 1970).

Keywords: RNA polymerase I (RNA聚合酶I)

Ribosomal RNA transcription (核糖体RNA转录)

Nuclei isolation (细胞核分离)

Radiolabelled uridine incorporation (放射性标记的尿苷结合)

α-amanitin (α-鹅膏蕈碱)

Background

In eukaryotic cells the RNA polymerase I transcribes ribosomal genes, which are located in the nucleolus, producing 45S rRNA precursor molecules. These are processed to form the mature 18S, 5.8S and 28S rRNA. They are essential for the assembly of the 60S and the 40S subunits of mature ribosomes. Recent evidence indicates that the ribosome biogenesis rate is related to cell cycle length (Derenzini et al., 2005) and may play a role in tumorigenesis by controlling the expression of the tumour suppressor protein p53. Cells with an up-regulated ribosome biogenesis are rapidly proliferating and are characterized by a down-regulated p53 expression (Donati et al., 2011). Moreover, the ribosome biogenesis rate influences the sensitivity of cancer cells to chemotherapeutic agents which hinder rRNA transcription: higher the rate of ribosome biogenesis, higher the cytotoxic effect induced by the drug (Scala et al., 2016). Therefore, the evaluation of the ribosome biogenesis rate will become a more and more utilized procedure both in tumour pathology and in clinical oncology (Montanaro et al., 2013). Since the rate of ribosome production is tightly conditioned by the rate of 45S precursor molecules transcription, all the methods used for the evaluation of ribosome biogenesis rate measure the synthesized 45S rRNA. The used methods are: quantitative evaluation of 45S rRNA transcripts by real time PCR analysis; quantitative analysis of 45S rRNA, separated by gel electrophoresis of total RNA extracted from cells labelled with ³²P-orthophosphate, and visualized by autoradiography; and quantitative evaluation of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I. The first two methods measure the quantity of 45S rRNA present in the cells, that may be influenced by changes of rRNA processing mechanism, whereas the method described here quantifies the transcriptional activity of the RNA polymerase I and it is indicative of the rRNA transcription rate. This method is very complex and time-consuming and requires special accuracy, but it is still the only one method to selectively measure the rRNA transcription rate.

Materials and Reagents

Cell scraper, 40 cm handle, 16 mm blade (Sigma-Aldrich, catalog number: C6106 )
Plastic tubes 50 ml conical base (114 x 28 mm) (SARSTEDT, catalog number: 62.547.254 )
Plastic tubes 15 ml conical base (120 x 17 mm) (SARSTEDT, catalog number: 62.554.002 )
Plastic tubes 1.5 ml safe lock (Eppendorf, catalog number: 022363204 )
Glass microfiber filters, diameter 25 mm, Whatman grade GF/C (GE Healthcare, catalog number: 1822-025 )
Scintillation plastic vials (Sigma-Aldrich, catalog number: V6755 )
Microscope slides (26 x 76 mm) (Sigma-Aldrich, catalog number: Z692247 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3264 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791 )
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
Tris base (Sigma-Aldrich, catalog number: T1503 )
Ammonium sulfate [(NH₄)₂SO₄] (Sigma-Aldrich, catalog number: A4418 )
Sucrose (Merck Millipore, catalog number: 107687 )
1 M magnesium chloride (MgCl₂) solution (Sigma-Aldrich, catalog number: M1028 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
1 M manganese(II) chloride (MnCl₂) solution (Sigma-Aldrich, catalog number: M1787 )
Guanosine 5’-triphosphate sodium salt (GTP) (Sigma-Aldrich, catalog number: 10106399001 )
Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A7699 )
Cytidine 5’-triphosphate disodium salt (CTP) (Sigma-Aldrich, catalog number: C1506 )
Uridine 5’-triphosphate tri salt (UTP) (Sigma-Aldrich, catalog number: U6875 )
Uridine 5’ triphosphate [5,6-³H] tetrasodium salt (³H-UTP) (PerkinElmer, catalog number: NET380250UC )
α-amanitin (Sigma-Aldrich, catalog number: A2263 )
Liquid scintillation cocktail Hionic Fluor (PerkinElmer, catalog number: 6013319 )
Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T4885 )
Perchloric acid (PCA) 65% w/w (10.35 N) (CARLO ERBA Reagents, catalog number: 306091 )
Potassium hydroxide (KOH) (EMD Millipore, catalog number: 105033 )
2-mercaptoethanol (EMD Millipore, catalog number: 805740 )
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
PBS (pH 7.3) (see Recipe 1)
1 M Tris-HCl solution (pH 7.4 or pH 8.0) (see Recipe 2)
3 M ammonium sulfate (see Recipe 3)
Homogenization-solution (see Recipe 4)
Washing solution (see Recipe 5)
Suspension solution (see Recipe 6)
High ionic strength 5x reaction mixture (see Recipe 7)
Nucleoside triphosphate mixture (see Recipe 8)

Non-radioactive solution
Radioactive solution

α-amanitin reconstitution (see Recipe 9)
10% (w/w) TCA (see Recipe 10)
0.6 N PCA (see Recipe 11)
0.3 N KOH (see Recipe 12)
6 N PCA (see Recipe 13)
7% (w/w) PCA (see Recipe 14)
0.5 M NaF (see Recipe 15)
Low ionic strength 5x reaction mixture (see Recipe 16)

Equipment

Protective gloves/protective clothing/eye protection/face protection
Centrifuge (Beckman Coulter, model: TJ-25 )
Note: This product has been discontinued.
Teflon^® pestle PYREX^® Potter-Elvehjem tissue grinder (Thomas Scientific, catalog number: 1234F )
Forceps
High speed tissue grinder (homogenizer) (IKA, model: Eurostar )
Inverted microscope (Zeiss, model: Primovert, catalog number: 491206-0001-000 )
10 ml glass tubes
Shaking water bath (JULABO, model: SW22 )
2,000 ml filter flask
Spectrophotometer UV-VIS (CHROMSERVIS, model: Nanogenius )
Liquid scintillation counter (Guardian, Wallac, PerkinElmer)
Vacuum glass filter holder for 25 mm disc filters elements: glass funnel, clamp, base for 25 mm glass/filter holder and a perforated stopper (available from many companies, for example: Sigma-Aldrich, catalog number: Z290467 ; EMD Millipore, catalog number: XX1002500 )
Vacuum pump