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Epicentre Forum 1 (2)
The 5´-termini of many natural RNA molecules, including
most eukaryotic messenger RNAs, viral RNAs, many small nuclear RNAs (snRNA)
and heterogeneous nuclear RNAs (hnRNA) have a structure called a "cap".
Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride
bonds in the triphosphate bridge of the cap structure, releasing the
cap nucleoside and generating a 5´-phosphorylated terminus on the
RNA molecule. The resulting "decapped" 5´-phosphorylated RNA can
then be manipulated for further studies. For example, intramolecular
ligation using RNA Ligase of a viral genomic RNA after treatment with
TAP has been used to determine the nucleic acid sequence of the 5´-
and 3´-termini.1 Procedures
involving ligation of oligoribonucleotides to TAP-treated RNAs followed
by amplification of the ligated product have been developed for mapping
the 5´-termini of mRNAs.2,3 Removal
of the cap structure using TAP also permits the RNA to be 5´-end
labeled for sequencing or for use as a hybridization probe. The TAP-treated
RNA is dephosphorylated with alkaline phosphatase, then labeled using
T4 Polynucleotide Kinase (PNK) and usually [gamma-32P]-ATP.
Table 1 presents a general TAP protocol for "decapping" RNA
and subsequently labeling the 5´-termini. Epicentre's TAP is certified
free of contaminating RNase, phosphatase and DNA exo- and endonuclease.
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Table 1.
"Decapping" RNA Using Tobacco Acid Pyrophosphatase
- Quantitate RNA by UV absorbance.
- Precipitate the RNA with ethanol; wash the resulting pellet
with 70% ethanol.
- Resuspend the RNA pellet to a final concentration of 1 µg/µl
in 50 µl 1X TAP Buffer.
- Add 1U TAP enzyme per pmol RNA; incubate at 37°C for 1
hr.
- Precipitate the "decapped" RNA with ethanol.
Important: Contrary to some reports, ATP is not required
for TAP activity and, in fact, will decrease "decapping" activity
by acting as a competitive substrate.
5´-End-Labeling "Decapped" RNA Using T4
PNK
- Dephosphorylate the TAP-treated RNA with alkaline phosphatase.
- Inactivate the phosphatase by phenol extraction.
- Precipitate the dephosphorylated RNA with ethanol.
- Resuspend the RNA in 2.5 µl 10X PNK Buffer (Epicentre).
- Add 0.5 µl [gamma-32P]-ATP, 5-10 U T4 Polynucleotide
Kinase (Epicentre) and RNase-free H2O to 25 µl
final volume.
- Incubate at 37°C for 1 hr.
- Inactivate the PNK by incubating the reaction mixture at 70°C
for 5 min.
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- C.W. Mandl, F.X. Heinz, E. Puchhammer-Stockl and C. Kunz (1991), Sequencing
the Termini of Capped Viral RNA by 5´'3´ Ligation and PCR,
BioTechniques 10:485-486.
- M. Fromont-Racine, E. Bertrand, R. Pictet and T. Grange (1993), A
Highly Sensitive Method for Mapping the 5´-Termini of mRNAs,
Nuc. Acids Res. 21:1683-1684.
- K. Maruyama and S. Sugano (1994), Oligo-Capping: a Simple Method
to Replace the Cap Structure of Eucaryotic mRNAs with Oligoribonucleotides,
Gene 138: 171-174.
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