Figure 2. Protection of precursor RNA from RNase T1 digestion by polyadenylation complex formation. Polyadenylation complexes were formed on the same RNA as in Figure 1 (20 min incubation) and treated with RNase T1 (see Protocol 3). (A) Two such complexes, U (upper) and L (lower), lane 3, were separated on a non-denaturing 4% polyacrylamide gel after RNase T1 digestion ( + ). The reason for the appearance of two specific complexes is unknown. Control complexes (lane 2) were not incubated with RNase T1 (-); 'pre' indicates precursor not exposed to nuclear extract. 'H' (lane 1) represents a non-specific complex (see Figure 1). (B) RNA oligonucleotides were purified from complexes U and L and analysed on a denaturing 12% polyacrylamide gel (C). The 67 nt oligonucleotide from U and L was eluted from the gel, digested with RNase T1 and analysed on a denaturing 20% polyacrylamide gel. The four fragments visible from U and L arise from both sides of the polyadenylation site. N (lane 1) shows RNase T1 digestion products from precursor RNA. The numbers indicate sizes (nt) of RNase T1 fragments or DNA size markers. Reproduced from ref. 13.

An estimation of the total binding site size from the sum of these secondary oligonucleotides may, therefore, not be possible.

Figure 2 shows an example of RNase T1 protection. In the case of the adenovirus L3 polyadenylation site used in this experiment, a fragment of 67 nt, containing the AAUAAA sequence as well as downstream sequences, is protected (.Figure 2B). A second RNase T1 digestion after purification of the protected 67 nt oligonucleotide generates several RNA fragments of between 6-21 nt, which lie on both sides of the polyadenylation site (Figure 2C). (The nucleotide sequence of the substrate RNA used in this experiment and its pattern of RNase T1 digestions products can be found in ref. 13.)

2.4.4 Analysis of polyadenylation complex formation by modification-interference

RNase protection yields only information about the segment of RNA that is stably bound by processing factors. In contrast, modification-interference analyses indicate which particular nucleotides are essential for the formation of processing complexes (15). In this procedure, end-labelled substrate RNA is either modified chemically at purine bases with diethylpyrocarbonate or pyrimidine bases are removed with hydrazine. As in nucleotide sequencing by the chemical method, the extent of modification must be very low, so that less than one base is modified per RNA molecule. The modified RNA is used to form processing complexes. After non-denaturing gel electrophoresis, complexed RNA is recovered from the gel. Both this RNA apd substrate RNA that has not been incubated with protein are then cleaved at the sites of chemical modification and the cleavage products are separated on a sequencing (denaturing) gel. The non-complexed RNA provides gel tracks containing purine-specific or pyrimidine-specific cleavage ladders with which the RNA from the polyadenylation complex can be compared. If the modification of any particular nucleotide has interfered with complex formation, the band corresponding to this nucleotide will be missing in the ladders generated from the RNA sample isolated from the complex.

The same procedure can be used to investigate the binding of purified processing factors, such as CPSF to AAUAAA-containing RNAs. It can also be applied to study the formation of any other RNA-protein complexes. The method is not even limited to experiments in which a complex formed between proteins and the modified RNA is separated by native gel electrophoresis. Other methods (e.g. denaturing gel electrophoresis) can also be used to isolate the RNA that is accepted as a substrate in the reaction of interest and to separate it from the RNA that is excluded.

The procedure for modification-interference (see Protocol 5) uses an RNA precursor labelled at its 3'-end by a ligation procedure (see Protocol 4),

Protocol 4. 3' end-labelling of RNA substrates by ligation

Equipment and reagents

• Denaturing 6% Polyacrylamide gel, electrophoresis equipment, TBE buffer, and formamide loading buffer (Volume I, Chapter 1, Protocol 8)

• Unlabelled capped substrate RNA synthesized as described in Volume i. Chapter 1, Protocol 4

• [5'-32P]pCp (New England Nuclear or Amersham; 3000Ci/mmol)

• 10 x RNA ligase buffer (0.5 M Tris-HCI, pH 7.9, 0.15 M MgCI2, 33 mM DTT3)

• 0.25 mM ATP, diluted from a stock solution (see Protocol 2)

1. In a microcentrifuge tube, mix 2 pi each of 0.25 mM ATP, 0.1 mg/ml BSA, 10 x RNA ligase buffer, and DMSO plus 10pi of [5'-32P]pCp.

2. Add 5-10 pmol of substrate RNA (equivalent to 80-160 ng of RNA 50 nt in length) in a volume of up to 2 pi.

3. Cool the mixture to 4 °C and add 10-20 units of RNA ligase.

5. Purify full-length labelled RNA by denaturing Polyacrylamide gel electrophoresis (see Volume I, Chapter 1, Protocol 8).

a Add DTT just before use from the 1.0 M stock.

Protocol 5. Analysis of polyadenylation complex formation by modification-interference

Equipment and reagents

• Non-denaturing polyacrylamide gel and electrophoresis buffer (see Protocol 2), 12% denaturing polyacrylamide gel (sequencing gel), TBE buffer, and form-amide loading buffer (Volume I, Chapter 1, Protocol 8) together with the appropriate electrophoresis equipment

• Electroblotting apparatus for wet blotting (e.g. Bio-Rad or Hoefer)

• DEAE transfer membrane (Schleicher and Schuell, NA45)

• HeLa cell nuclear extract and reagents for generating polyadenylation complexes (see Protocol 2)

• 3' end-labelled substrate RNA (see Protocol 4)

• 1.0M sodium acetate, adjusted to pH 4.5 with acetic acid

• 50mM sodium acetate, pH4.5, 1 mM EDTA, use the 1 M sodium acetate stock to make this solution without adjusting the pH

• 0.3 M sodium acetate, adjusted to pH 3.8 with acetic acid

• Diethylpyrocarbonate (DEPC) (Sigma)3

• Anhydrous hydrazine (Eastman-Kodak)6 containing 0.5 M NaCI, prepare this solution by dissolving solid NaCI in hydrazine

• 1.0 M aniline in 0.3 M sodium acetate, pH 3.8, use redistilled aniline15

• TBE buffer containing 0.1% SDS (see Protocol 3)

• NA45 elution buffer (55% formamide", 1.8 M sodium acetate, 2 mM EDTA, 0.2% SDS), use solid sodium acetate, not a solution whose pH has been adjusted

• Urea loading buffer (8.0 M urea, 20 mM Tris-HCI, pH 7.9, 1 mM EDTA, 0.05% xylene cyanol, 0.05% bromophenol blue)

Preparation of RNA substrates

1. Use precursor RNA as the substrate, synthesized without radioactive label as described in Volume I, Chapter 1, Section 2.2, and 3' end-labelled as Protocol 4. Mix two separate aliquots of this RNA (approximately 106c.p.m. each) with 12.5 pg tRNA as carrier. Add 0.1 vol. of 7.5 M ammonium acetate and 2.5 vol. of absolute ethanol. Vortex and chill the mixture on ice for a few minutes.

2. Centrifuge the mixture for 1 5 min in a microcentrifuge. Discard the supernatant. In this and all subsequent steps, control the recovery of RNA with a hand-held Geiger counter.

3. Add 1 ml of 70% ethanol to the pellet. Centrifuge for 5 min in a microcentrifuge. Discard the supernatant and dry the pellet under vacuum.

Purine modification

4. Dissolve one RNA sample in 200 pi of 50 mM sodium acetate, pH 4.5, 1 mM EDTA.

5. Add 3 pi of fresh DEPC and incubate the RNA for 5 min at 90 °C with the lid of the tube open.

6. Add 75 pi of 1 M sodium acetate, pH 4.5, and 900 mI of ethanol. Collect the RNA precipitate as in steps 1 and 2.

7. Dissolve the RNA pellet in 200 mI of 0.3 M sodium acetate, pH 3.8. Precipitate the RNA by adding 600 mI of ethanol. Collect the RNA precipitate as in steps 1 and 2.

8. Dissolve the RNA in 10 pi of water. Store it at -20 °C.

Pyrimidine modification

9. Dissolve the other RNA sample (steps 1-3) in 20 m' of anhydrous hydrazine containing 0.5 M NaCI and incubate it for 70 min at 4 °C.

10. Add 200 Ml of 0.3 M sodium acetate, pH 3.8, and 900 m> ethanol, then collect the RNA precipitate as in steps 1 and 2.

11. Dissolve and reprecipitate the RNA pellet as in step 7.

12. Dissolve the RNA in 10 |jl of water and store it at -20 °C.

Complex formation and electrophoresis

13. Set aside one-tenth of each sample of modified RNA (steps 8 and 12) as control samples for later aniline cleavage and electrophoresis steps. Use the rest of each sample separately for complex formation as described in Protocol 2B but use a reaction volume of 1 25 m'-

14. Load each sample on the non-denaturing gel using several adjacent lanes to accommodate the entire sample.

15. Run the gel as in Protocol 2C.

16. After electrophoresis, transfer the RNAe onto an NA45 DEAE membrane by electroblotting in a wet blotting cell. To do this, cut out one piece of NA45 membrane slightly larger than the gel and soak it for 10 min in 10mM EDTA, pH 7.6, at room temperature.

17. Transfer the membrane into 0.5 M NaOH and let it soak for 5min.

18. Wash the membrane in 5 changes of water (2 min each).

19. Cut out four pieces of Whatman 3MM paper of the same size as the NA45 membrane and wet them in TBE buffer containing 0.1% SDS.

20. Place the NA45 membrane onto a stack of two pieces of 3MM paper. Place the gel directly onto the DEAE membrane and finally two sheets of 3MM paper on top. Be careful not to trap air bubbles.

21. Assemble this sandwich in the blotting cell according to the manufacturer's instructions as for Western blotting. For transfer, use TBE buffer containing 0.1% SDS. Blot for 6 h in a coldroom at 50 V.

22. After the transfer is complete, wrap the DEAE membrane in cling film to prevent it drying and expose it to X-ray film using guide marks to align the membrane on the film (see Protocol 3, step 9).

23. Cut out strips from the membrane corresponding to the specific polyadenylation complex.

24. Elute the RNA from the DEAE membrane by soaking the membrane strips in NA45 elution buffer for 1 5 min at 70 °C.

25. Remove the elution buffer from the membrane into fresh tubes. Add 12.5 pg carrier tRNA to each RNA sample and extract once with an equal volume of phenol:chloroform.

26. Add 2.5 vol. of ethanol. Collect the RNA precipitate and wash it as in steps 1 -3.

27. Purify full-length RNA on a denaturing polyacrylamide gel (see Volume I, Chapter 1, Protocol 8).

Cleavage of modified RNA

28. Dissolve each of the modified RNAs from steps 27 in 20 pi of 1 M aniline in 0.3 M sodium acetate, pH 3.8. Incubate for 20 min at 60 °C in the dark. Similarly treat samples of the control RNAs not used to form polyadenylation complexes (step 13).

30. Vortex and recover the RNA precipitate by centrifugation in a microcentrifuge for 1 5 min.

31. Redissolve the RNA in 1 50 pi of 1 % SDS and repeat steps 29 and 30.

32. Wash the RNA pellets by adding 1 ml of 70% ethanol and centrifuging in a microcentrifuge for 5 min.

33. Dissolve the RNA in 5 pi of urea loading buffer.

34. Determine the amount of radioactivity recovered by Cerenkov counting.

35. Dilute aliquots of each sample (containing equal amounts of radioactivity) with urea loading buffer, heat for 90 sec at 90 °C and load on a 12% denaturing polyacrylamide gel (sequencing gel)'.

36. Run the gel until the bromophenol blue marker is 1 5 cm from the bottom and detect the RNA by autoradiography.

a Store opened bottles of DEPC in a refrigerator for no more than one week. b Store hydrazine in a closed container with some desiccating material in a fume hood at room temperature.

c Redistill the aniline under nitrogen and store it in aliquots at - 20 °C in the dark. Be careful: aniline is toxic and combustible.

d Deionize the formamide with a mixed-bed ion-exchange resin and store it in the dark at -20 °C.

" The extent to which proteins contained in the complex are transferred to the DEAE membrane together with the RNA is unknown. Any protein transferred to the membrane and eluted in steps 21-24 will be removed by the phenol:chloroform extraction in step 25.

1 If the sample in urea loading buffer is heated for too long, urea may precipitate. Redissolve it by adding a small amount of water.

A few experimental points should be noted with respect to the modification-interference procedure (.Protocol 5):

(a) The procedure does not tolerate much non-specific degradation of RNA since this will generate bands in the sequence ladder that do not stem from aniline cleavage of modified nucleotides. Therefore, exercise care to avoid RNase contamination (see Volume I, Chapter 1, Section 1.1). The shorter a substrate RNA is the easier it is to use in this protocol since short molecules are obviously less susceptible to non-specific degradation than long molecules. RNase inhibitor from placenta may also be used (see Protocol 6, footnote b).

(b) The RNA substrate used for modification-interference can also be 5 ' end-labelled with [-y-32P] ATP and polynucleotide kinase instead of with RNA ligase. However, it has been reported that 5'-labelled RNA does not give clean sequencing ladders with pyrimidine modifications (15).

(c) If, after analysis, the extent of modification is found to be too high or too low, vary the incubation times for the modification reactions (see Protocol 5, steps 5 and 9).

(d) In Protocol 5, the sequence ladders to which the RNA from the specific polyadenylation complex is compared are generated from substrate RNA that has never been incubated with nuclear extract. These control ladders may also be generated from RNA isolated from non-specific complexes. For unknown reasons, cleavage at G residues is more efficient with RNA that has been incubated with extract and eluted from the non-denaturing gel. The best control is thus to use both substrate RNA without incubation and RNA from non-specific complexes.

Figure 3 shows the results of a modification-interference experiment that was carried out with purified CPSF rather than nuclear extract. The nucleotide sequence of the substrate RNA can be read in the outer lanes (uncomplexed

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