Vladimir Pena

Structure of a multipartite protein-protein interaction domain in splicing factor Prp8 and its link to retinitis pigmentosa

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.

Structure and function of an RNase H domain at the heart of the spliceosome

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.

Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

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.

Common Design Principles in the Spliceosomal RNA Helicase Brr2 and in the Hel308 DNA Helicase

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 C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction

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 × 104. 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.

Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome

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.

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.

Crystal structure of a DNA catalyst.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. 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 fold. Here we report the crystal structure of the RNA-ligating 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.

The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes

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. Aquariuss 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.

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.