Experimental Verification of the Secondary Structures of Guide RNA-Pre-mRNA Chimaeric Molecules in Trypanosoma Brucei

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Abstract
RNA editing in kinetoplastid organisms is an RNA-processing reaction that adds and deletes U nucleotides at specific sites in mitochondrial pre-mRNAs. The edited sequence is specified by guide RNAs and the processing presumably occurs within a high-molecular-mass ribonucleoprotein complex containing several enzymatic activities. Although the mechanism is not currently known, potential intermediates or by-products of the editing process are chimaeric RNAs where guide (g) RNAs are covalently attached, via their non-encoded U-tail, to their cognate pre-mRNAs. We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure-sensitive chemical and enzymatic probes. The experiments revealed a bipartite domain structure consisting of a gRNA/pre-mRNA interaction hairpin and an independently folding mRNA stem/loop in all three RNAs. The connecting U-tail was a determinant for the length of the interaction stems with the oligo(U) nucleotides base pairing to internal gRNA sequences. The probed structures have calculated delta G27o values of -92 kJ/ mol to -134 kJ/mol, somewhat less stable than the predicted minimal free energy structures and support previously proposed models for the interaction between gRNAs and pre-mRNAs. Optical melting studies indicated additional, higher order structural features for all three molecules with four defined melting transition between 10 degrees C and 90 degrees C. A comparison of CD spectra in the absence and presence of mitochondrial protein extracts demonstrated no gross structural changes of the RNA structures induced by the association with polypeptides.