Structural basis for specific RNA recognition by the alternative splicing factor RBM5.

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10.

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex.

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3'-end processing machineries.

Deciphering mechanistic implications of antimicrobial and antioxidant potentials of certain new dibutyltin(IV) formulations as possible therapeutic options based on DFT and hybrid materials paradigm.

Mechanistic implications of antimicrobial and in vitro antioxidant potentials of a set of newly generated nonbridged mononuclear N,O-orthometallated and carboxylate bridged binuclear nonorthometallated dibutyltin(IV) formulations have been investigated. Some of these formulations were screened for their antibacterial and antifungal activities against Escherichia coli and Candida albicans, respectively whereas in vitro antioxidant potential was examined by Ferric reducing antioxidant power (FRAP) assay. Nonbridged mononuclear N,O-orthometallated dibutyltin(IV) formulations were generated by the reactions of Bu2 SnCl2 with sodium salts of 2-aminophenol/substituted 2-aminophenol and flexible N-protected amino acids in 1:1:1 molar ratio in refluxing dry THF. Plausible structures of these nonbridged mononuclear N,O-orthometallated dibutyltin(IV) formulations containing flexible N-protected amino acids have been suggested on the basis of spectroscopic and mass studies of some representative formulations. Plausible structures suggested on the basis of spectroscopic studies are corroborated by density functional theory (DFT/B3LYP method) (SPARTAN-20) investigation of a representative dibutyltin(IV) complex and the ligands involved in it. The presence of two different classes of organic ligands in this complex provides an opportunity to study optimized topologies, bonding, distortions, optimized energy, and stability of the complex.

The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain.

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation.

The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly 'closed' conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an 'open' Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

Conformational Dynamics from Ambiguous Zinc Coordination in the RanBP2-Type Zinc Finger of RBM5.

The multi-domain RNA binding protein RBM5 is a molecular signature of metastasis. RBM5 regulates alternative splicing of apoptotic genes including the cell death receptor Fas and the initiator Caspase-2. The RBM5 RanBP2-type zinc finger (Zf1) is known to specifically recognize single-stranded RNAs with high affinity. Here, we study the structure and conformational dynamics of the Zf1 zinc finger of human RBM5 using NMR. We show that the presence of a non-canonical cysteine in Zf1 kinetically destabilizes the protein. Metal-exchange kinetics show that mutation of the cysteine establishes high-affinity coordination of the zinc. Our data indicate that selection of such a structurally destabilizing mutation during the course of evolution could present an opportunity for functional adaptation of the protein.

Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions.

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαß) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP-RNP interactions in general.

Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.

Structural basis for the recognition of spliceosomal SmN/B/B' proteins by the RBM5 OCRE domain in splicing regulation.

The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique ß-sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B'. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B'. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B' proteins in FAS/CD95 alternative splicing regulation.

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets ß-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-ß surface of ß-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the ß-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.

The binding modes and binding affinities of epipodophyllotoxin derivatives with human topoisomerase IIα.

Epipodophyllotoxin derivatives have important therapeutic value in the treatment of human cancers. These drugs kill cells by inhibiting the ability of topoisomerase II (TP II) to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. The 3D structure of human TP IIα was modeled by homology modeling. A virtual library consisting of 143 epipodophyllotoxin derivatives has been developed. Their molecular interactions and binding affinities with modeled human TP IIα have been studied using the docking and Bimolecular Association with Energetics (eMBrAcE) developed by Schrödinger. Structure activity relationship models were developed between the experimental activity expressed in terms of percentage of intracellular covalent TP II-DNA complexes (log PCPDCF) of these compounds and molecular descriptors like docking score and free energy of binding. For both the cases the r2 was in the range of 0.624-0.800 indicating good data fit and r2(cv) was in the range of 0.606-774 indicating that the predictive capabilities of the models were acceptable. Low levels of root mean square error for the majority of inhibitors establish the docking and eMBrAcE based prediction model as an efficient tool for generating more potent and specific inhibitors of human TP IIα by testing rationally designed lead compounds based on epipodophyllotoxin derivatization.