A

OH OH CHO CHO

Protocol 5. Chemical decapping by Reagents

• Capped cellular mRNA (~100pg/ml) purified as described in Volume I, Chapter 1, Section 3

• Metaperiodate reagent (freshly prepared, 10 mM Nal04 in 0.15 M sodium acetate, pH 5.3)

ixidation and (3-elimination reaction

• TSE buffer (20 mM Tris-HCI, pH 7.4, 0.5 mM EDTA, 0.5% SDS)

• Carrier yeast tRNA (Sigma) dissolved in H20 at 2 mg/ml

Method

Periodate oxidation

1. Mix the capped cellular mRNA (- 1 pg) with 20 pg of carrier tRNA in 10 pi H20.

2. Add 1 pi of periodate reagent and incubate the mixture on ice for 1 h in the dark.

3. Add 10 pi of 20% glycerol to stop the oxidation.

4. Precipitate the RNA by adding 0.15 ml of 2.0 M ammonium acetate, pH ~ 7.0 and 0.85 ml of cold (- 20 °C) ethanol.

6. Centrifuge the tube at 10 000gr for 15min at 4 °C. Carefully remove and discard the supernatant.

7. Redissolve the RNA pellet in 0.18 ml TSE buffer. Add 0.18 ml of 2.0 M ammonium acetate, pH -7.0, followed by 0.9 ml ethanol.

8. Repeat steps 6 and 7.

9. Dry the RNA pellet under reduced pressure and redissolve it in 20 pi of TSE buffer.

/?-elimination reaction

10. Add 0.1 ml of 0.3 M aniline, pH 5.3, mix and incubate the mixture for 5 min at 30 °C.

Yasuhiro Furuichi and Aaron J. Shatkin Protocol 5. Continued

11. Precipitate the RNA by adding 0.1 ml of 2.0 M ammonium acetate, pH -7.0, and 0.55 ml of cold (-20 °C) ethanol.

12. Recover the RNA as described in steps 5-8.

13. Redissolve the decapped RNA in 10 pi of distilled H20.

3.2.2 Recapping mRNA

The decapped triphosphorylated RNA is recapped by incubation with the vaccinia virus capping enzyme complex which contains RNA triphosphatase and methyltransferase(s) as described in Protocol 6. The reaction sequence is:

where * and x denote 32P- and [3HI methyl-labelled positions, respectively.

Protocol 6. Recapping mRNA with [a-32P]GTP Reagents

• Decapped mRNA (~0.5-1.0pg in ~5-10m' H20) prepared as in Protocol 5"

• Guanylyltransferase from vaccinia virus (Gibco-BRL catalogue # 8024SA, 1 -5 U/pl)

Method

1. Set up the recapping reaction by mixing the following reagents in a

microcentrifuge tube on ice:

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