The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryoEM structures of the yeast E complex assembled on introns, providing the first view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, which either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyzes back-splicing generating circRNA (on long exons). The model is supported by our experiments demonstrating that E complex assembled on the yeast EFM5 or HMRA1 middle exon can be chased into circRNA when the exon is sufficiently long. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and ILS complexes revealed mechanisms of 5′ splice site (ss) recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release. Here, we report a cryoEM structure of the post-catalytic P complex at 3.3Å resolution, revealing that 3′ ss is mainly recognized through non-Watson-Crick basepairing with the 5′ ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3′ exon and potentially regulating Prp22 activity. Prp22 binds nucleotides 15–21 in the 3′ exon, enabling it to pull the intron-exon or ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading or exon release, respectively.
Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.
Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A, a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.
Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.
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