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Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated Bact and the branching-competent B* spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16)1–3. However, because PRP2 is observed only at the periphery of spliceosomes3–5, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic6,7. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate—the BAQR complex—found halfway through the catalytic activation process. The cryogenic electron microscopy structure of BAQR reveals how PRP2 and Aquarius remodel Bact and BAQR, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)—a complex chaperoning the selection of branch and 3′ splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept., Chemical modulation of intron selection has emerged as a route for cancer therapy. Here, structures of the U2 snRNP’s SF3B module and of prespliceosome- both in complexes with splicing modulators- provide insight into the mechanisms of intron recognition and branch site inactivation.

Cohesin is a protein complex whose core subunits, Smc1, Smc3, Scc1, and SA1/SA2 form a ring-like structure encircling the DNA. Cohesins play a key role in the expression, repair, and segregation of eukaryotic genomes. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate the cohesin subunit Smc3, thereby inducing stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute cohesin as tri- or tetrameric assemblies and use those as physiologically-relevant substrates for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the murine Esco2 active site cleft, are critical for catalysis. Our experiments support a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, also involving two nearby serine residues - S566 and S527- that possess a proton relay function.

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bact complex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5′ splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile—the branchsite adenosine—is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5′ss. Our structure suggests that Prp2 adenosine triphosphatase–mediated remodeling leads to conformational changes in Hsh155’s HEAT domain that liberate the first-step reactants for catalysis.

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 For further resources related to this article, please visit the WIREs website.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes)1 or synthetic genetic polymers2. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage3. DNA-catalysed reactions include RNA and DNA ligation in various topologies4,5, hydrolytic cleavage6,7 and photorepair of DNA8, as well as reactions of peptides9,10 and small molecules11,12. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold13. Here we report the crystal structure of the RNAligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms. peerReviewed

SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.

Summary 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 SF3b155's 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 SF3b155's 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.

Proceedings of the National Academy of Sciences of the United States of America 109, 17418-17423 (2012). doi:10.1073/pnas.1208098109, Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, involves multiple RNA-protein remodeling steps, driven by eight conserved DEXD/H-box RNA helicases. The 250-kDa Brr2 enzyme, which is essential for U4/U6 di-small nuclear ribonucleoprotein disruption during spliceosome catalytic activation and for spliceosome disassembly, is the only member of this group that is permanently associated with the spliceosome, thus requiring its faithful regulation. At the same time, Brr2 represents a unique subclass of superfamily 2 nucleic acid helicases, containing tandem helicase cassettes. Presently, the mechanistic and regulatory consequences of this unconventional architecture are unknown. Here we show that in human Brr2, two ring-like helicase cassettes intimately interact and functionally cooperate and how retinitis pigmentosa-linked Brr2 mutations interfere with the enzyme's function. Only the N-terminal cassette harbors ATPase and helicase activities in isolation. Comparison with other helicases and mutational analyses show how it threads single-stranded RNA, and structural features suggest how it can load onto an internal region of U4/U6 di-snRNA. Although the C-terminal cassette does not seem to engage RNA in the same fashion, it binds ATP and strongly stimulates the N-terminal helicase. Mutations at the cassette interface, in an intercassette linker or in the C-terminal ATP pocket, affect this cross-talk in diverse ways. Together, our results reveal the structural and functional interplay between two helicase cassettes in a tandem superfamily 2 enzyme and point to several sites through which Brr2 activity may be regulated., Published by Academy, Washington, DC

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 spliceosome's 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 spliceosome's catalytic centre into an active conformation.

Summary Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19—a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays in vitro , and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways.

Brr2 is a unique DExD/H box protein required for catalytic activation and disassembly of the spliceosome. It contains two tandem helicase cassettes that both comprise dual RecA-like domains and a noncanonical Sec63 unit. The latter may bestow the enzyme with unique properties. We have determined crystal structures of the C-terminal Sec63 unit of yeast Brr2, revealing three domains, two of which resemble functional modules of a DNA helicase, Hel308, despite lacking significant sequence similarity. This structural similarity together with sequence conservation between the enzymes throughout the RecA-like domains and a winged helix domain allowed us to devise a structural model of the N-terminal active cassette of Brr2. We consolidated the model by rational mutagenesis combined with splicing and U4/U6 di-snRNA unwinding assays, highlighting how the RecA-like domains and the Sec63 unit form a functional entity that appears suitable for unidirectional and processive RNA duplex unwinding during spliceosome activation and disassembly.

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.

Protein Prp8 interacts with several other spliceosomal proteins, snRNAs, and the pre-mRNA and thereby organizes the active site(s) of the spliceosome. The DEAD-box protein Brr2 and the GTPase Snu114 bind to the Prp8 C terminus, a region where mutations in human Prp8 are linked to the RP13 form of Retinitis pigmentosa. We show crystallographically that the C-terminal domain of yeast Prp8p exhibits a Jab1/MPN-like core known from deubiquitinating enzymes. Insertions and terminal appendices are grafted onto this core, covering a putative isopeptidase center whose metal binding site is additionally impaired. Targeted yeast-two-hybrid analyses show that the RP13-linked region in the C-terminal appendix of human Prp8 is essential for binding of human Brr2 and Snu114, and that RP13 point mutations in this fragment weaken these interactions. We conclude that the expanded Prp8 Jab1/MPN domain represents a pseudoenzyme converted into a protein-protein interaction platform and that dysfunction of this platform underlies Retinitis pigmentosa.

Aquarius is a multifunctional putative RNA helicase that binds precursor-mRNA introns at a defined position. Here we report the crystal structure of human Aquarius, revealing a central RNA helicase core and several unique accessory domains, including an ARM-repeat domain. We show that Aquarius is integrated into spliceosomes as part of a pentameric intron-binding complex (IBC) that, together with the ARM domain, cross-links to U2 snRNP proteins within activated spliceosomes; this suggests that the latter aid in positioning Aquarius on the intron. Aquarius's ARM domain is essential for IBC formation, thus indicating that it has a key protein-protein-scaffolding role. Finally, we provide evidence that Aquarius is required for efficient precursor-mRNA splicing in vitro. Our findings highlight the remarkable structural adaptations of a helicase to achieve position-specific recruitment to a ribonucleoprotein complex and reveal a new building block of the human spliceosome.

Full-length A beta 1-42 and A beta 1-40, N-truncated pyroglutamate A beta 3-42 and A beta 4-42 are major variants in the Alzheimer brain. A beta 4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of A beta 4-x and pyroglutamate A beta 3-X mitigated neuron loss in Tg4-42 mice expressing A beta 4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of A beta 4-42. NT4X reduced pyroglutamate A beta 3-x, A beta x-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. A beta 1-x and A beta x-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated A beta starting with position four in addition to pyroglutamate A beta 3-x is a relevant target to fight Alzheimer's disease.

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.

Precursor-messenger RNA (pre-mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre-mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8-specific elements. Comparisons to RNase H–substrate complexes suggested how an RNA encompassing a 5′-splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre-mRNAs and snRNAs. A truncated RNase H-like active centre lies next to a known contact region of the 5′SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of splicing.

The protein giant neurofibromin (320kDa) is the protein product of the NF1 tumor suppressor gene, alterations of which are responsible for the pathogenesis of neurofibromatosis type 1 (NF1). Neurofibromin is a Ras-specific GTPase activating protein (RasGAP) that, 15 years after the cloning of the gene, remains the only clearly defined function of the protein. In a structural proteomics approach, we aimed at defining functions beyond RasGAP activity based on the discovery of structural modules. Given the poor outcome of domain prediction tools, we have undertaken a fragment solubility survey covering the full protein sequence, with the aim of defining new domain boundaries or fragments that could be investigated by biochemical methods including structural analysis. More than 200 constructs have been expressed and tested for solubility in small scale assays. Boundaries were chosen based upon secondary structure predictions, sequence conservation among neurofibromin orthologues and chemical properties of amino acids. Using this strategy we recently discovered a novel bipartite module in neurofibromin. We have expanded our study to include ESPRIT, a library-based construct screen, to perform fragment testing at a finer level with respect to the choice of terminal residues. Our study confirms earlier notions about the challenges neurofibromin presents to the biochemist and points to strategies whereby the success rate may be enhanced in the future.

Beyond participating in splicing, some spliceosomal components from higher eukaryotes play pivotal roles in coupling splicing to other RNA-processing events. The massive spliceosomal helicase Aquarius (170 kDa) is loaded at a specific location on the intron, where it acts as a molecular linker between splicing, the deposition of the exon-junction complex on the mRNA and the formation of snoRNPs from intronic segments. We have determined the crystal structures of the full-length Aquarius in complex with a non-hydrolysable ATP analog as well as with a single-stranded RNA. The structure reveals a complex molecular architecture, with a central core that resembles the RNA helicase Upf1 surrounded by several specific domains. Among them we identify an ARM-repeat domain, plus three other domains that were not observed in helicases. We could evidence that Aquarius possesses motor activity in vitro and that, strikingly, it exhibits opposite unwinding polarity versus Upf1. Crystal structure of Aquarius in complex with RNA revealed a novel mode of RNA binding by a helicase and led to the identification of structural adaptations underlying the reversal of directionality. Overall, the structure-function analysis brings insightful implications into the manner how two helicases have diverged from a common ancestor as a mean to achieve opposite polarities of translocation. Furthermore, we demonstrate that Aquarius is recruited to the spliceosome in complex with four other splicing factors and that the ARM domain is essential for the integrity of the complex. Moreover,the ARM domain makes contacts in the spliceosomal core, providing insight into the molecular basis underlying the accurate positioning of Aquarius on the intron. Following the line of these findings, we finally show that Aquarius plays an important role for the occurrence of splicing per se.