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Nucleic Acids Research Pages 3449-3450  

Chemical mapping of co-existing RNA structures
Acknowledgement
References

Chemical mapping of co-existing RNA structures

Chemical mapping of co-existing RNA structures

Astrid R. W. Schröder, Tilman Baumstark, Detlev Riesner*

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany

Received April 17, 1998; Revised and Accepted June 3, 1998

ABSTRACT

In many cases RNA can assume co-existing or meta-stable structures preventing structure determination by chemical mapping. A novel method is described, by which RNA is modified with dimethyl sulphate without shifting the distribution of different structures. The different structures are then separated in native gel electrophoresis, and structure determination by primer extension can be carried out separately for each structure.

Chemical and enzymatic probing is widely used and easy to apply for investigation of RNA structures (for reviews see 1,2). The RNA structure which is present in solution under physiological conditions can be identified experimentally among a number of potential structures that might be predicted from sequence data, free energy calculations and phylogenetic studies. Dimethyl sulphate (DMS) reacts as a chemical probe with nucleophilic centres of heterocyclic bases and thereby leads to detection of those adenosines and cytidines in vitro and in vivo which are not involved in Watson-Crick interactions. In order to interpret correctly the chemical and enzymatic mapping results, it was necessary up to now to establish experimental conditions in which the RNA under study will assume only one single structure and retain it throughout the experiment. If the structure of interest is the most thermodynamically stable structure, heating followed by slow cooling guarantees the RNA to assume that structure. However, in many cases an RNA can adopt different co-existing structures under native conditions and some of these might even be metastable, i.e. they are not in the state of lowest free energy, but are stable for a limited time with a biological activity.

One example for a biologically active metastable RNA structure is the replication control of plasmid Col E1 by duplex formation between antisense RNA 1 and the primer RNA, which can assume several metastable intermediates, and only one of these intermediates is accessible to RNA 1 (3,4). Another example comes from a study on splicing in HeLa cell extracts, where the kinetics of RNA folding can play a role in alternative splicing (5). Our group is interested in structure and function of viroid RNA and reported that for viroid replication (6), as well as for viroid processing (7,8), metastable RNA structures are important.

Given the importance of metastable RNA structures in vivo, it is of outstanding interest to analyse different metastable structures by chemical mapping if they co-exist under native conditions. This was impossible with the known mapping protocols because they required the establishment of a single RNA structure as described above. Thus in every single case, a very special protocol has to be elaborated to establish the respective single metastable structure without knowing its kinetic and thermodynamic features.

Here we present a rapid method, combining temperature-gradient gel electrophoresis (TGGE) (9) and DMS probing, which allowed us to analyse co-existing and/or metastable RNA structures without the difficulties mentioned above. The example presented in this study is the potato spindle tuber viroid (PSTVd), which belongs to the smallest plant pathogens, consists only of a single-stranded circular RNA molecule and is replicated autonomously in the host cell via a rolling circle mechanism (for review see 10). It was shown that a longer-than-unit RNA transcript of a PSTVd-sequence can be processed to mature circular viroids in a nuclear extract, and that only one out of four possible structures of this RNA is an active substrate in this reaction. It was obvious from an analysis by TGGE of the RNA after in vitro transcription, that under these particular conditions at least two different structures co-existed (Fig. 1A). The active structure ExMTL is metastable and contains a GNRA tetraloop, whereas the thermodynamically most stable structure (ExL) is inactive (7). We had to develop sophisticated protocols to establish each of the structures separately (7) and found after chemical mapping of each of the structures, two main differences at positions C102 and C262 which confirmed our secondary structure models presented in Figure 1B (8).


Figure 1. Structural analysis of a PSTVd longer-than-unit length plus stranded in vitro transcript (TB110), which is processable in a nuclear extract (7,8). (A) TGGE analysis of the co-existing structures ExM and ExL, generated by in vitro transcription. A linear temperature increase was established perpendicular to the electric field. The instrument was a Qiagen TGGE system (Qiagen, Hilden, Germany), now commercially available from Biometra (Göttingen, Germany). Electrophoresis was carried out at 300 V for 10 min at homogeneous temperature of 15°C and for 1.5 h in the presence of the gradient from 20 to 60°C, using 5% polyacrylamide, 0.17% bisacrylamide and 0.2× TBE (1× 89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA). Further details were described earlier (7). (B) Secondary structure models of the structural motifs of ExM and ExL differing only in the central part of the characteristic rod-like structure of viroids with the terminal parts identical to circular PSTVd indicated by straight lines left and right (8). Bases characteristic for the respective motif are given in outlined typeface; stars indicate non-canonical base-pairs (G [star] A, reversed Hoogsteen A [star] U, A [star] A). The most important differences in DMS modification pattern are denoted by rectangles; filled broad rectangles indicate strong accessibility, filled narrow rectangles moderate accessibility and open rectangles protection against DMS modification, respectively.

Using this RNA we tried to develop a procedure for the simultaneous mapping of co-existing structures, instead of establishing the unique structures and carrying out several chemical mappings. One should note that experimental probing conditions had to be found under which both structures remained stable and the conversion of the metastable into the most stable structure during the mapping procedure was prevented.

Several incubation times and buffer conditions were tested and the stability of our two model structures was controlled by TGGE. Best results were obtained when chemical modification of the co-existing structures was carried out in a 200 µl volume containing 50 mM HEPES-KOH pH 8.0, 1 mM EDTA, 3 µg RNA transcript containing the co-existing structures, 7 µg tRNA and 0.1% DMS for 30 min on ice. The reaction was stopped by addition of 14 µg carrier tRNA and subsequent dialysis against 0.2× TBE buffer at 4°C for 1 h. Then the methylated RNA structures could be separated on a native PAA-gel at a temperature where the two RNA structures have different gel electrophoretic mobilities, i.e. 25°C (Fig. 1A), as derived from TGGE. Each of the bands was isolated by elution from the gel (11), and the respective modification sites were identified separately by primer extension analysis of 50 ng eluted RNA as described previously (8).

The data obtained from simultaneous modification of the co-existing structures in four independent experiments (Fig. 2, lanes CO) are in good accordance with the data obtained previously after pre-establishing of the single structures (Fig. 2, lanes PE) (8); for example C102 is accessible to DMS in ExL and protected in ExM according to both experimental procedures (Fig. 2).


Figure 2. Section of the DMS modification pattern of the viroid transcript pTB110 in structure ExM and ExL (cf. Fig. 1B). The transcript was pre-treated to establish either structure ExM or ExL, modified and analysed separately (lanes PE) or both structures were modified simultaneously under conditions where both structures co-exist, separated on a gel and analysed after gel extraction (lanes CO). Lane 4, transcript pTB110 treated only with buffer. The modification sites were separately identified by primer extension and analysed on an 8% PAA denaturing gel in comparison to the viroid sequence in plasmid DNA (lanes A, C, G and T, respectively). The most important difference in modification pattern of ExL and ExM is designated by an arrow.

The results show that different conformers of one and the same RNA can be probed simultaneously by chemical mapping without working out detailed protocols to establish a unique structure in solution. It has been assumed tacitly, and was confirmed by the present results, that chemical modification itself does not change the structure; neither the gel electrophoretic mobility nor the thermodynamic stability. This was expected, since chemical modification affects the non-base paired nucleotides and therefore does not alter the basepairing scheme; modified and non-modified RNA molecules migrate in the same band. Up to now, our understanding of metastable and co-existing structures was limited by secondary structure calculations and experimental analysis of TGGE. The method described here adapts the technique of chemical mapping to the problem of co-existing and metastable structures and might widen our view of unique RNA structures to structure ensembles.

ACKNOWLEDGEMENT

The work was supported by grants from the Deutsche Forschungsgemeinschaft.

REFERENCES

1. Ehresmann,C., Baudin,F., Mougel,M., Romby,P., Ebel,J.-P. and Ehresmann,B. (1987) Nucleic Acids Res., 15, 9109-9128. MEDLINE Abstract

2. Kolchanov,N.A., Titov,I.I., Vlassova,I.E. and Vlassov,V.V. (1996) Prog. Nucleic Acid Res. Mol. Biol., 53, 132-196.

3. Polinsky,B., Zhang,X.-Y. and Fitzwater,T. (1990) EMBO J., 9, 295-304.

4. Gultyaev,A.P., van Batenburg,F.H.D. and Pleij,C.W.A. (1995) J. Mol. Biol., 250, 37-51. MEDLINE Abstract

5. Eperon,L.P., Graham,J.R., Griffiths,A.D. and Eperon,I.C. (1988) Cell, 54, 393-401. MEDLINE Abstract

6. Qu,F., Heinrich,C., Loss,P., Steger,G., Tien,P. and Riesner,D. (1993) EMBO J., 12, 2129-2139. MEDLINE Abstract

7. Baumstark,T. and Riesner,D. (1995) Nucleic Acids Res., 23, 4246-4254. MEDLINE Abstract

8. Baumstark,T., Schröder,A.R.W. and Riesner,D. (1997) EMBO J., 16, 599-610. MEDLINE Abstract

9. Rosenbaum,V. and Riesner,D. (1989) Biophys. Chem., 26, 235-246.

10. Diener,T.O. (ed.) (1987) The Viroids. Plenum Publishing Corporation,New York.

11. Krupp,G. (1988) Gene, 72, 75-89. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +49 211 811 4840; Fax: +49 211 811 5167; Email: riesner{at}biophys.uni-duesseldorf.de

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