Modified nucleotides - impact on RNA structure and function

The project presented here is concerned with naturally occuring modified nucleotides in RNA. Up to date more than 90 differently modified nucleotides are known in cellular RNAs, however their structural and functional role is far from being well understood and will be addressed in the two topics of this proposal. First, based on our recently introduced codon-anticodon pairing model (Angew. Chem. 2000, 39, 922-926), we intend to investigate the influence of conserved modified tRNA nucleotides within - and next to - the anticodon sequence on the pairing properties of the corresponding codon-anticodon core duplex. The effects arising from these nucleoside modifications concern among others nucleobase stacking, steric repulsion and metal ion coordination; in general, all of them modulate the stability of RNA double helixes. The thermodynamic pairing parameters of triplet RNA interactions are tractable by means of our cyclic (and open-chain) model compounds and provide a solid basis to directly correlate them to a variety of decoding phenomena in context with the accuracy during ribosomal translation; one subtopic will target the modified tRNA purine bases at position 37 in order to investigate if these nucleotides exert a balancing effect on the pairing strength of cognate` triplets with different sequence composition. Another subtopic will target frameshift sites in order to investigate if thermodynamic preferences are an intrinsic property of these conserved nucleotide sequences. We aim to contribute towards a better understanding of the molecular details of codon-anticodon interactions. This first part of the proposal represents the scientific continuation and expansion of the soon ending project P13216-CHE. The second topic is a new development and hits the question how modified nucleotides affect RNA folding. With respect to secondary and tertiary structure modified nucleosides are generally related to modulator influences meaning that they attenuate and amplify preexisting structures. In preliminary studies, we have first evidence that a certain class of modifications, namely nucleosides that are methylated at the Watson Crick pairing site of their nucleobases, possess the potential to substantially affect RNA structure by mediating the folding into different secondary structure motifs. Our approach will be based on the site specific incorporation of methylated nucleobases into oligoribo-nucleotide sequences that form duplexes if unmethylated but fold into hairpins if methylated. We aim to correlate the sequence prerequisites, the specific methylation sites of nucleobases and the position of the methylated nucleoside within a sequence that are responsible to trigger changes in RNA secondary structure. In a second stage we will expand these model investigations on biological relevant methylated sequence motives encountered in tRNAs, the ribosome and other cellular RNAs.

The original overall scientific goal of the project was a comprehensive investigation of the influence of naturally occurring nucleoside modifications on RNA structure and function. The major results can be summerized as following: First, we investigated the impact of methylated nucleosides on RNA structure and folding. In this concern, a significant outcome was that changes of RNA secondary structure can be efficiently induced by nucleosides that are methylated at the Watson-Crick base pairing site, provided a thermodynamically favorable base pairing alternative is encountered in the particular sequence. We also suggested that this concept has been realized in nature, for instance, in the small ribosomal subunit comprising a double helical stem that is capped by a hypermethylated loop. Using short RNA model strands, we found strong evidence that the methylation pattern therein is crucial for the fold observed and prevents the 3`-terminal 16S rRNA sequence from misfolding. Second, we learned from the comprehensive study mentioned above on naturally occurring nucleoside methylations that these nucleosides can be held responsible for the selection of a defined RNA secondary structure out of several alternatives. We used this knowledge to develop artificial nucleoside modifications with the aim to create chemical tools to trigger RNA folding processes. A proof of principle was achieved with so called trichloroethyl guanosine modified RNA. This concept might be of high relevance for the development of novel riboregulators that can be be applied for control of gene expression. Third, we also initiated studies on selenium-modified RNA. We established important steps towards their reliable chemical and enzymatic synthesis. Selenium-modified RNA represents a tool to facilitate phasing of RNA X-ray crystallographic data. This development originates from our deep interest in understanding RNA structure and function and has forged a very fruitful cooperation with a leading structural biologist. It was the basis for the first structure determination of a ribozyme that catalyzes carbon-carbon bond formation.