Topology of the U12-U6atac snRNA complex of the minor spliceosome and binding by NTC-related protein RBM22

Abstract

ABSTRACTSplicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly composed of five small nuclear (sn)RNAs and >100 proteins. RNA components catalyze the two transesterification reactions, but proteins perform critical roles in assembly and rearrangement. The catalytic core comprises a paired complex involving U2 and U6 snRNAs for the major form of the spliceosome and U12 and U6atac snRNAs for the minor variant (~0.3% of all spliceosomes in higher eukaryotes); the latter performs identical chemistry, despite limited sequence conservation outside key catalytic elements, and lack of the multi-stem central junction found in the U2-U6 snRNA complex. Here we use solution NMR techniques to show that base pairing patterns of the U12-U6atac snRNA complex of both human and Arabidopsis share key elements with the major spliceosome’s U2-U6 snRNA complex; probing of the single-stranded segment opposing termini of the snRNAs indicates elongation in this region in place of the stacked base pairs at the base of the U6 intramolecular stem loop in the U2-U6 snRNA complex. Binding affinity of RBM22, a protein implicated in remodeling human U2-U6 snRNA prior to catalysis, to U12-U6atac was analyzed by electrophoretic mobility shift assays in which we monitored migration of both protein and RNA components in the same gel. Results indicate that RBM22 binds the U2-U6 and U12-U6atac snRNA complexes specifically and with Kd = 3.5 µM and 8.2 µM, respectively. Similar affinity between RBM22 and each RNA complex suggests that the protein performs the same role in both spliceosomes.