Jana Schmitzová

Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.

The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.

Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosomes catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.

Molecular Architecture of SF3b and Structural Consequences of Its Cancer-Related Mutations

SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155s HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155s HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.

Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre

RNA‐structural elements play key roles in pre‐mRNA splicing catalysis; yet, the formation of catalytically competent RNA structures requires the assistance of spliceosomal proteins. We show that the S. cerevisiae Cwc2 protein functions prior to step 1 of splicing, and it is not required for the Prp2‐mediated spliceosome remodelling that generates the catalytically active B* complex, suggesting that Cwc2 plays a more sophisticated role in the generation of a functional catalytic centre. In active spliceosomes, Cwc2 contacts catalytically important RNA elements, including the U6 internal stem‐loop (ISL), and regions of U6 and the pre‐mRNA intron near the 5′ splice site, placing Cwc2 at/near the spliceosomes catalytic centre. These interactions are evolutionarily conserved, as shown by studies with Cwc2s human counterpart RBM22, indicating that Cwc2/RBM22–RNA contacts are functionally important. We propose that Cwc2 induces an active conformation of the spliceosomes catalytic RNA elements. Thus, the function of RNA–RNA tertiary interactions within group II introns, namely to induce an active conformation of domain V, may be fulfilled by proteins that contact the functionally analogous U6‐ISL, within the spliceosome.

The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome

Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2s NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2s RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2s ATPase activity to remodeling of the spliceosome into a catalytically active machine.

Dynamic contacts of U2, RES, Cwc25, Prp8 and Prp45 proteins with the Pre-mRNA branch-site and 3' splice site during catalytic activation and step 1 catalysis in yeast spliceosomes.

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast Bact spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the Bact crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosomes protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA‐binding protein

The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosomes catalytic centre. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV‐induced crosslinking of Cwc2–U6 snRNA allowed the identification by mass spectrometry of six RNA‐contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the Torus and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA‐binding platform to bring RNA elements of the spliceosomes catalytic centre into an active conformation.

The organization and contribution of helicases to RNA splicing.

Splicing is an essential step of gene expression. It occurs in two consecutive chemical reactions catalyzed by a large protein–RNA complex named the spliceosome. Assembled on the pre‐mRNA substrate from five small nuclear proteins, the spliceosome acts as a protein‐controlled ribozyme to catalyze the two reactions and finally dissociates into its components, which are re‐used for a new round of splicing. Upon following this cyclic pathway, the spliceosome undergoes numerous intermediate stages that differ in composition as well as in their internal RNA–RNA and RNA–protein contacts. The driving forces and control mechanisms of these remodeling processes are provided by specific molecular motors called RNA helicases. While eight spliceosomal helicases are present in all organisms, higher eukaryotes contain five additional ones potentially required to drive a more intricate splicing pathway and link it to an RNA metabolism of increasing complexity. Spliceosomal helicases exhibit a notable structural diversity in their accessory domains and overall architecture, in accordance with the diversity of their task‐specific functions. This review summarizes structure–function knowledge about all spliceosomal helicases, including the latter five, which traditionally are treated separately from the conserved ones. The implications of the structural characteristics of helicases for their functions, as well as for their structural communication within the multi‐subunits environment of the spliceosome, are pointed out. WIREs RNA 2016, 7:259–274. doi: 10.1002/wrna.1331

Emerging views about the molecular structure of the spliceosomal catalytic center.

Pre-mRNA splicing occurs in two chemical steps that are catalyzed by a large, dynamic RNA-protein complex called the spliceosome. Initially assembled in a catalytically inactive form, the spliceosome undergoes massive compositional and conformational remodeling, through which disparate RNA elements are re-configured and juxtaposed into a functional catalytic center. The intricate construction of the catalytic center requires the assistance of spliceosomal proteins. Recent structure-function analyses have demonstrated that the yeast-splicing factor Cwc2 is a main player that contacts and shapes the catalytic center of the spliceosome into a functional conformation. With this advance, corroborated by the atomic structure of the evolutionarily related group IIC introns, our understanding of the organization and formation of the spliceosomal catalytic center has progressed to a new level.