Various polyamine dendrimers with a triethanolamine core inhibit the activity of the Candida ribozyme by forming RNA-dendrimer complexes via electrostatic interactions.

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.DOI: http://dx.doi.org/10.7554/eLife.23533.001

The RNA helicase Prp2 facilitates the remodeling of the spliceosomal B complex to the catalytically activated B* complex just before step one of splicing. As a high-resolution cryo-EM structure of the B* complex is currently lacking, the precise spliceosome remodeling events mediated by Prp2 remain poorly understood. To investigate the latter, we used chemical structure probing to compare the RNA structure of purified yeast B and B* complexes. Our studies reveal deviations from conventional RNA helices in the functionally important U6 snRNA internal stem-loop and U2/U6 helix Ib in the activated B complex, and to a lesser extent in B*. Interestingly, the N7 of U6-G60 of the catalytic triad becomes accessible to DMS modification in the B* complex, suggesting that the Hoogsteen interaction with U6-A52 is destabilized in B*. Our data show that Prp2 action does not unwind double-stranded RNA, but enhances the flexibility of the first step reactants, the pre-mRNA's 5' splice site and branch site adenosine. Prp2 therefore appears to act primarily as an RNPase to achieve catalytic activation by liberating the first step reactants in preparation for catalysis of the first step of splicing.

The precise function of the trimeric retention and splicing (RES) complex in pre-mRNA splicing remains unclear. Here we dissected the role of RES during the assembly and activation of yeast spliceosomes. The efficiency of pre-mRNA splicing was significantly lower in the absence of the RES protein Snu17, and the recruitment of its binding partners, Pml1 (pre-mRNA leakage protein 1) and Bud13 (bud site selection protein 13), to the spliceosome was either abolished or substantially reduced. RES was not required for the assembly of spliceosomal B complexes, but its absence hindered efficient B complex formation. ΔRES spliceosomes were no longer strictly dependent on Prp2 activity for their catalytic activation, suggesting that they are structurally compromised. Addition of Prp2, Spp2, and UTP to affinity-purified ΔRES B or a mixture of B/B complexes formed on wild-type pre-mRNA led to their disassembly. However, no substantial disassembly was observed with ΔRES spliceosomes formed on a truncated pre-mRNA that allows Prp2 binding but blocks its activity. Thus, in the absence of RES, Prp2 appears to bind prematurely, leading to the disassembly of the ΔRES B complexes to which it binds. Our data suggest that Prp2 can dismantle B complexes with an aberrant protein composition, suggesting that it may proofread the spliceosome's RNP structure prior to activation.

Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional folding at a rate constant similar to the folding of the active ribozyme, and folding of both P3-P7 and P1-P10 pseudoknots are inhibited by antisense oligonucleotides. We conclude that when RNA folding is coupled with transcription, formation of pseudoknot structures dominates the productive folding pathway and serves as a rate-limiting step in producing the self-splicing competent Candida intron.