B

Figure 1. Time course of the formation of polyadenylation complexes and polyadenylated RNA. Polyadenylation complexes were formed according to Protocol 2 on an RNA (246 nt) containing the adenovirus L3 polyadenylation site. (A) Complexes were analysed on a non-denaturing 4% polyacrylamide gel. For an explanation of the complexes formed, see the text. Precursor RNA is indicated by 'pre'. A control lane with RNA in the absence of nuclear extract is labelled '-NXT'. (B) RNA was purified from aliquots of the same samples as in (A) and analysed on a 6% denaturing gel. Reproduced from ref. 13.

In the procedure described in Protocol 2, RNA can be purified from a second aliquot of each sample and analysed on a denaturing gel so that a time course of the processing reaction itself is obtained (for a detailed discussion, see Section 2.5.2 and Protocol 6). Although complex formation is very rapid, polyadenylated product appears only after a significant lag period (compare Figures 1A and IB). The RNA present in each of the various complexes can be analysed as described (ref. 13; see also Section 2.4.3, Protocol 3).

2.4.3 Analysis of polyadenylation complexes by RNase protection

RNA sequences involved in the formation of the polyadenylation complex are protected by the binding of processing factors against digestion with RNase Tl.

This nuclease cleaves RNA on the 3 '-side of GMP residues, producing a pattern of oligonucleotides that is characteristic for every RNA and that can be predicted from its sequence.

In Protocol 3, the polyadenylation complexes are treated with a low concentration of RNase T1 and then separated on a non-denaturing gel. After the polyadenylation complexes have been isolated from the gel, they are deproteinized and their protected RNA fragments are further analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. In the final stage of the procedure, each of these fragments is isolated from the denaturing gel and subjected to a second RNase T1 digestion and denaturing gel electrophoresis. RNase T1 cleavage sites that were protected during the first digestion will be accessible during the second digestion. A comparison of the oligonucleotides generated from the protected fragments during the second digestion to the pattern of oligonucleotides generated from the naked substrate RNA will identify those sites that were protected during the first digestion and thus the regions of the RNA involved in polyadenylation complex formation.

The polyadenylation complex is generated as described in Protocol 2. However, for the efficient detection of RNA fragments after the various RNase digestions, the RNA substrate must have a higher specific radioactivity.

Protocol 3. Analysis of polyadenylation complex formation by RNase T1 protection

Equipment and reagents

• Non-denaturing Polyacrylamide gel and electrophoresis equipment (see Protocol 2)

•Two denaturing polyacrylamide (sequencing) gels®, one with 12% acryl-amide and the other with 20%, TBE running buffer, and formamide loading buffer (see Volume I, Chapter 1, Protocol 8)

•RNase T1 (Calbiochem). Make a stock solution (50 units/nD in 10 mM Tris-HCI, pH 7.5, 1 mM EDTA and dilute this in the same buffer as required

•Proteinase K (10mg/ml; Boehringer Mannheim)

•RNA elution buffer (0.75 M ammonium acetate, 10 mM magnesium acetate, 1 % (v/v) phenol, 0.1 % SDS, 25 ng/ml tRNA)

• 32P-labelled RNA substrate (20 000 c.p.m./fmol)'', dialysed HeLa cell nuclear extract, and reagents for generating the polyadenylation complex (see Protocol 2)

• TBE buffer (25 mM Tris base, 25 mM boric acid, 1 mM EDTA) containing

• TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA) containing 2.5 mg/ml tRNA and 1 unit/pi RNase T1

• 32P-labelled DNA electrophoresis size markers covering the range between 20 and 100 nt (e.g. pBR322 digested with HpaW and end-labelled with [7-32P]ATP and polynucleotide kinase; see Volume

1. Set up a 25 pi polyadenylation reaction as in Protocol 2B. To do this, dispense approximately 107c.p.m. of labelled precursor RNA into a microcentrifuge tube.

2. Depending on the way the RNA has been stored, centrifuge or lyophilize the RNA in a microcentrifuge tube as described in Protocol 2B, steps 2 or 3. In this and all subsequent steps, control the recovery of RNA with a hand-held Geiger counter.

4. Vortex to dissolve the RNA. PLace the tube on ice and add 5 pi of 12.5% PVA and 12.5pl HeLa cell nuclear extract.

5. Mix gently by pipetting and incubate for 30min at 30 °C.

6. Add 0.1 unit of RNase T1 and incubate the mixture for 10 min at room temperature.

7. Add 25 mg/ml heparin to 0.2 mg/ml final concentration.

8. Incubate for 10 min at room temperature then load an aliquot (at least 5 pl) or all of the sample onto the non-denaturing polyacrylamide gel. Separate the complex by electrophoresis as in Protocol 2C.

9. After electrophoresis, wrap the gel in cling film. Place an X-ray film on the gel and use a fibre-tip pen to apply several guide marks extending from the X-ray film to the cling film. Expose and develop the film.

10. Place the gel on top of the autoradiograph and use the marks to align the film. Using the autoradiographic image as a guide, and using a clean scalpel, cut out a gel piece containing the specific polyadenylation complexc.

11. Soak the gel piece in TBE buffer containing 0.1% SDS for 10 min at room temperature, then incubate the gel slice for 30 min at 30 °C in TBE buffer containing 0.1% SDS and 0.5 mg/ml proteinase K.

12. Using clean forceps, place the gel piece in a well of the 12% denaturing polyacrylamide gel.

13. Mix radioactive DNA size markers with formamide loading buffer. Heat the solution for 3 min to 95 °C, chill it on ice, and load it into one of the wells of the denaturing gel.

14. Run the gel until the bromophenol blue marker has reached the bottom of the gel.

15. Autoradiograph the gel and cut out gel pieces containing the major protected RNA fragment(s) as in step 10.

• 0.2 M creatine phosphate

2 pi

16. Incubate each gel fragment separately overnight at 37 °C in 400 pi of RNA elution buffer.

17. Remove the buffer from the gel fragments into a fresh microcentrifuge tube.

18. Extract the eluted RNA by vortexing with an equal vol. of phenol:chloroform.

19. Centrifuge in a microcentrifuge for 30 sec to separate the phases.

20. Remove the upper (aqueous) phase into a fresh microcentrifuge tube. Add 2.5 vol. of ethanol, mix, and centrifuge for 1 5 min in a microcentrifuge.

21. Discard the supernatant. Add 1 ml of 70% ethanol to the tube and centrifuge it again for 5 min. Discard the supernatant and dry the pellet under vacuum.

22. Dissolve the RNA oligonucleotide in 4 pi of TE buffer containing 2.5 mg/ml tRNA and 1 unit/pl of RNase T1. Similarly set up a sample of the original RNA substrate for RNase T1 digestion as a control.

23. Digest the samples for 30 min at 30 °C.

24. Lyophilize each sample and then dissolve them in formamide loading buffer. Boil each for 3 min and chill on ice.

25. Load the samples onto the 20% denaturing polyacrylamide gel. Run the gel until the bromophenol blue marker is 10 cm from the bottom.

26. Autoradiograph the gel.

aThe 12% gel should be of the same thickness as the native gel (1 mm) so that gel fragments from the native gel can be placed on top of the denaturing gel. The 20% gel should be a normal (thin) sequencing gel.

6 Make RNA of the required specific activity by using the appropriate specific activity of [a-32P]UTP in the transcription reaction. Obviously, the intensity of the autoradiographic bands in the gels (steps 9, 1 5, and 26) depends on the number of UMP residues in each RNase T1 fragment. Fragments containing no UMP residues will not be visible. It may thus be more convenient to label the RNA with [a-32P]GTP. Since every fragment contains only a single GMP residue at its 3'-end, this will result in uniform labelling intensity of all fragments.

cThe RNase T1 digestion may change the position of the specific complexes in the gel. Non-specific complexes may disappear.

The RNase T1 digestion pattern of an RNA can be predicted from its sequence and most of the larger digestion products can be identified unambiguously by their size. Thus, the protected RNA fragment obtained from the polyadenylation complex can also be identified by the pattern of oligonucleotides generated from it in the second RNase T1 digestion. If a fragment obtained in this second digestion cannot be identified by its size alone, an additional digestion, e.g. with RNase T2, may be used to determine its nucleotide composition (14).

The size of the protected fragment before the second digestion gives an estimate of the total size of the binding site. This is significant because the distribution of GMP residues in the substrate RNA may not allow the identification of every oligonucleotide produced in the second digestion.

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