Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3′-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3′-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3′-end processing complexes that interact with the U-rich or U/G-rich elements at 3′-end cleavage/polyadenylation sites.
To achieve more potent and selective inhibition of disease‐causing proteins, we have generated PROTACs, or PROteolysis TArgetting Chimera. PROTACs are small, hetero‐bifunctional small molecules which simultaneously bind target proteins and E3 ubiquitin Ligases. Upon formation of a ternary complex, the Cullin‐based RING E3 ligase catalyzes the ubiquitination of the target protein, leading to its degradation. PROTACs have shown efficacy against a variety of targets in tissue culture and animal models, but mechanistic questions remain. Specifically, a prediction of the event‐based nature of PROTAC function is an ability to catalytically inhibit the target protein (i.e. the induction of supra‐stoichiometric ubiquitination of that target protein with respect to PROTAC). To rigorously define this catalysis, we have reconstituted the E1/E2/Cullin‐RING E3 ubiquitination reaction, and have shown that PROTACs are indeed capable of modifying supra‐stoichiometric amounts of target protein. Furthermore, as the ubiquitination system is presented with a non‐natural substrate, we hope to use this system to address questions surrounding lysine‐selection and ubiquitin chain‐type formation.
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