Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the S. cerevisiae catalytic center, we characterize factors that modulate function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 allele. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and the second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Rearrangements within the U6 snRNA Core during the Transition between the Two Catalytic Steps of Splicing.

The RNA catalytic core of spliceosomes as visualized by cryoelectron microscopy (cryo-EM) remains unchanged at different stages of splicing. However, we demonstrate that mutations within the core of yeast U6 snRNA modulate conformational changes between the two catalytic steps. We propose that the intramolecular stem-loop (ISL) of U6 exists in two competing states, changing between a default, non-catalytic conformation and a transient, catalytic conformation. Whereas stable interactions in the catalytic triplex promote catalysis and their disruptions favor exit from the catalytic conformation, destabilization of the lower ISL stem promotes catalysis and its stabilization supports exit from the catalytic conformation. Thus, in addition to the catalytic triplex, U6-ISL acts as an important dynamic component of the catalytic center. The relative flexibility of the lower U6-ISL stem is conserved across eukaryotes. Similar features are found in U6atac and domain V of group II introns, arguing for the generality of the proposed mechanism.