EXPERIMENTAL-VERIFICATION OF THE SECONDARY STRUCTURES OF GUIDE RNA-PRE-MESSENGER-RNA CHIMERIC MOLECULES IN TRYPANOSOMA-BRUCEI

RNA editing in kinetoplastid organisms is an RNA-processing reaction t hat adds and deletes U nucleotides at specific sites in mitochondrial pre-mRNAs. The edited sequence is specified by guide RNAs and the proc essing presumably occurs within a high-molecular-mass ribonucleoprotei n complex containing several enzymatic activities. Although the mechan ism is not currently known, potential intermediates or by-products of the editing process are chimaeric RNAs where guide (g) RNAs are covale ntly attached, via their non-encoded U-tail, to their cognate pre-mRNA s, We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure-sensitive che mical and enzymatic probes. The experiments revealed a bipartite domai n structure consisting of a gRNA/pre-mRNA interaction hairpin and an i ndependently 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 G(27)(0) values of -92 kJ/mol to -134 kJ/mol, somewhat less stable than the predicted minimal free e nergy structures and support previously proposed models for the intera ction between gRNAs and pre-mRNAs. Optical melting studies indicated a dditional, higher order structural features for all three molecules wi th four defined melting transitions between 10 degrees C and 90 degree s C. A comparison of CD spectra in the absence and presence of mitocho ndrial protein extracts demonstrated no gross structural changes of th e RNA structures induced by the association with polypeptides.