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Topology of the U12–U6atac snRNA Complex of the Minor Spliceosome and Binding by NTC-Related Protein RBM22
Splicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly including 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...
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Published in: | ACS omega 2020-09, Vol.5 (37), p.23549-23558 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Online Access: | Get full text |
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Summary: | Splicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly including 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 of 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 shares key catalytic sequence elements and performs identical chemistry. Here we use solution NMR techniques to show that the U12–U6atac snRNA complex of both human and Arabidopsis maintain base-pairing patterns similar to those in the three-helix model of the U2–U6 snRNA complex that position key elements to form the spliceosome’s active site. However, in place of the stacked base pairs at the base of the U6 snRNA intramolecular stem loop and the central junction of the U2–U6 snRNA complex, we see altered geometry in the single-stranded hinge region opposing termini of the snRNAs to enable interaction between the key elements. We then use electrophoretic mobility shift assays and fluorescence assays to show that the protein RBM22, implicated in remodeling the human U2–U6 snRNA complex prior to catalysis, also binds the U12–U6atac snRNA complexes specifically and with similar affinity as to U2–U6 snRNA (a mean K d for the two methods = 3.4 and 8.0 μM for U2–U6 and U12–U6atac snRNA complexes, respectively), suggesting that RBM22 performs the same role in both spliceosomes. |
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ISSN: | 2470-1343 2470-1343 |
DOI: | 10.1021/acsomega.0c01674 |