Date of Degree


Document Type


Degree Name





Nancy L. Greenbaum

Committee Members

Akira Kawamura

Sebastién Poget

Subject Categories

Other Biochemistry, Biophysics, and Structural Biology | Polymer Chemistry


minor spliceosome, U12-U6atac snRNA complex, NMR, EMSA, RBM22


Splicing of precursor messenger (pre-m)RNA is a critical process in eukaryotes in which the non-coding regions, called introns, are removed and coding regions, or exons, are ligated to form a mature mRNA. This process is catalyzed by the spliceosome, a multi-mega Dalton ribonucleoprotein complex assembled from five small nuclear ribonucleoproteins (snRNP) in the form of small nuclear (sn)RNA-protein complexes (U1, U2, U4, U5 and U6) and >100 proteins. snRNA components catalyze the two transesterification reactions while proteins perform critical roles in assembly and rearrangement. U2 and U6 snRNAs are the only snRNAs directly implicated in catalyzing the splicing of pre-mRNA. However, assembly and rearrangement steps prior to catalysis require numerous proteins. 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 minor spliceosome shares key catalytic sequence elements with the major spliceosome and performs identical chemistry. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations in equilibrium, characterized by four and three helices surrounding a central junction. The four-helix conformer is strongly favored in the in vitro protein-free state, while the three-helix conformer predominates in spliceosomes. The minor spliceosome does not exhibit the conformational heterogeneity in the junction found in the major spliceosome. 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 elongation in the single stranded hinge region opposing termini of the snRNAs to enable interaction between the key elements. Electrophoretic mobility shift assays and fluorescence assays show that the human spliceosomal protein RBM22, implicated in remodeling the human U2-U6 snRNA complex prior to catalysis but not yet definitively identified in minor spliceosomes, binds the U12-U6atac snRNA complexes specifically and with similar affinity as to U2-U6 snRNA (a mean Kd for the two methods = 3.4 μM and 8.0 μM for U2-U6 and U12-U6atac snRNA complexes, respectively), suggesting that RBM22 performs the same role in both spliceosomes; the small but reproducible difference in the measured affinities may be due to differences in binding of the junction and hinge regions of the U2-U6 and U12-U6atac snRNA complexes, respectively. These findings contribute to our overall understanding of the formation of the RNA catalytic core of the major and minor versions of the spliceosome. The absence of the central junction in the human U12-U6atac snRNA may help explain its somewhat lesser affinity for the protein and the slower rate of catalysis exhibited by the minor spliceosome than its major counterpart, suggesting this region also forms a recognition site with the protein.