Because of the extensive literature available on RNA nucleotide modification, this review has focused on such cases where significant structural changes of RNA conformation were observed as a consequence of nucleotide modification. It is important to understand the impact of nucleotide modification on RNA structure, since structure mediates all RNA function. This underscores once more the artificial character of the distinction between ‘functional’ and ‘structural’ modifications. These terms had initially been defined in this review in order to separate functional effects, mediated by direct contact of modified nucleotides with other molecules, from effects mediated by modification-induced conformational changes. As has ecome obvious, most evidence for a role of nucleotide modification in RNA folding has come from studies of tRNA, which maintain their structure without the help of proteins. Evidence is accumulating that thermodynamic and metabolic stabilization of classical tRNA is performed by the ensemble of ‘structural’ modifications, but that the effect of any single modification is not critical. This is in agreement with the fact that critical importance of single modifications have so far only been documented for tRNAs which deviate at least to some extent from the classical pattern shown in Figure 2B. One of these, initiator 
from yeast, has opened a new area of research, which is likely to show that expression and stability of tRNA may be more strictly controlled by modification events than hitherto assumed. Transfer RNAs, as the most heavily modified RNAs, thus still have a lot of secrets left to be discovered, while the structural impact of nucleotide modification in other RNAs is only starting to unveil.
The example of rRNA (116) illustrates well the problems one has to face when trying to investigate a modification-induced structural rearrangement in a large RNA, which actually has little physiological relevance when devoid of proteins. The X-ray structures of the ribosomal subunits have indeed revealed that the role of ribosomal proteins is mainly one of structural support for the RNA (67,120). One might thus speculate that nucleotide modification as an ancient remedy for RNA misfolding has been replaced to a certain degree by proteins, especially in those RNAs which today function as RNP.
At a time were small RNAs with new functions are discovered on a daily basis, the recent discovery of the involvement of pseudouridine formation in gene regulation might represent the tip of an iceberg (19), and post-transcriptional modification may turn out to be a factor of more general importance in gene expression than hitherto assumed. And why should modification-induced structural rearrangement not be involved? Advances in chemical synthesis of modified nucleotides and the rapidly progressing identification of enzymes involved in RNA modification will certainly lead to more detailed insights in the near future.
ACKNOWLEDGEMENTS
The author acknowledges funding by the DFG grant HE 3397/3-1. The author would like to thank Henri Grosjean, Jaunius Urbonavicius, Roberto Fiammengo and the referees for extremely helpful comments on such a vast topic. The author apologizes to those colleagues whose work could not be cited here. The Open Access publication charges for this article were waived by Oxford University Press.
Conflict of interest statement. None declared.
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