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Zusammenfassung:
The active 3D conformation of the spliceosome's catalytic U2/U6 RNA core is stabilised by a network of secondary and tertiary RNA interactions, but also depends on spliceosomal proteins for its formation. To determine the contribution towards splicing of specific RNA secondary and tertiary interactions in the U2/U6 RNA core, we introduced mutations in critical U6 nucleotides and tested their effect on splicing using a yeast in vitro U6 depletion/complementation system. Elimination of selected RNA tertiary interactions involving the U6 catalytic triad, or deletions of the bases of U6-U80 or U6-A59, had moderate to no effect on splicing, showing that the affected secondary and tertiary interactions are not required for splicing catalysis. However, removal of the base of U6-G60 of the catalytic triad completely blocked splicing, without affecting assembly of the activated spliceosome or its subsequent conversion into a B*-like complex. Our data suggest that the catalytic configuration of the RNA core that allows catalytic metal M1 binding can be maintained by Protein-RNA contacts. However, RNA stacking interactions in the U2/U6 RNA core are required for productive coordination of metal M2. The functional conformation of the U2/U6 RNA core is thus highly buffered, with overlapping contributions from RNA-RNA and Protein-RNA interactions.
