Judith Ebert

Molecular insights into the interaction of PYM with the Mago–Y14 core of the exon junction complex

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago–Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago–Y14. Here we show that PYM is a cytoplasmic RNA‐binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago–Y14 by means of its N‐terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense‐mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD.

Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex.

In mammals, Up-frameshift proteins (UPFs) form a surveillance complex that interacts with the exon junction complex (EJC) to elicit nonsense-mediated mRNA decay (NMD). UPF3b is the component of the surveillance complex that bridges the interaction with the EJC. Here, we report the 3.4 Å resolution crystal structure of a minimal UPF3b-EJC assembly, consisting of the interacting domains of five proteins (UPF3b, MAGO, Y14, eIF4AIII, and Barentsz) together with RNA and adenylyl-imidodiphosphate. Human UPF3b binds with the C-terminal domain stretched over a composite surface formed by eIF4AIII, MAGO, and Y14. Residues that affect NMD when mutated are found at the core interacting surfaces, whereas differences between UPF3b and UPF3a map at peripheral interacting residues. Comparison with the binding mode of the protein PYM underscores how a common molecular surface of MAGO and Y14 recognizes different proteins acting at different times in the same pathway. The binding mode to eIF4AIII identifies a surface hot spot that is used by different DEAD-box proteins to recruit their regulators.

Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4

The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.

Conformational Variability of Nucleo-cytoplasmic Transport Factors

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin β-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin β, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin β, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo‐atomic models of the ten‐subunit Saccharomyces cerevisiae exosome in the unbound and RNA‐bound states. In the RNA‐bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate‐threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome‐like complexes from all domains of life, and might have been present in the most recent common ancestor.

Determinants for Aurora-A activation and Aurora-B discrimination by TPX2.

The mitotic kinases Aurora-A and Aurora-B have similar amino-acid sequences but are differently localised and regulated during cell division. The basis for their interactions with different and specific regulators is unclear. Surprisingly, our recent structural studies indicate that TPX2 regulates Aurora-A activity by binding at a site that is conserved almost completely on Aurora-B. Here we investigate molecular determinants of TPX2-Aurora-A recognition. Using structure-based mutagenesis, we show that a single amino-acid difference on the surface of the kinase catalytic domain is key to the precision with which TPX2 discriminates between Aurora-A and Aurora-B. The conservation at this amino-acid position suggests that this discriminatory mechanism is likely to be conserved in higher eukaryotes.

Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13

Mago and Y14 are core components of the exon junction complex (EJC), an assembly central to nonsense-mediated mRNA decay in humans and mRNA localization in flies. The Mago-Y14 heterodimer shuttles between the nucleus, where it is loaded onto specific mRNAs, and the cytoplasm, where it functions in translational regulation. The heterodimer is imported back into the nucleus by Importin 13 (Imp13), a member of the karyopherin-beta family of transport factors. We have elucidated the structural basis of the Mago-Y14 nuclear import cycle. The 3.35 A structure of the Drosophila Imp13-Mago-Y14 complex shows that Imp13 forms a ring-like molecule, reminiscent of Crm1, and encircles the Mago-Y14 cargo with a conserved interaction surface. The 2.8 A structure of human Imp13 bound to RanGTP reveals how Mago-Y14 is released in the nucleus by a steric hindrance mechanism. Comparison of the two structures suggests how this unusual karyopherin might function in bidirectional nucleocytoplasmic transport.

Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex.

Summary The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.

The Structure of the Asap Core Complex Reveals the Existence of a Pinin-Containing Psap Complex

The ASAP complex interacts with the exon-junction complex (EJC), a messenger ribonucleoprotein complex involved in post-transcriptional regulation. The three ASAP subunits (Acinus, RNPS1 and SAP18) have been individually implicated in transcriptional regulation, pre-mRNA splicing and mRNA quality control. To shed light on the basis for and consequences of ASAPs interaction with the EJC, we have determined the 1.9-Å resolution structure of a eukaryotic ASAP core complex. The RNA-recognition motif of RNPS1 binds to a conserved motif of Acinus with a recognition mode similar to that observed in splicing U2AF proteins. The Acinus–RNPS1 platform recruits the ubiquitin-like domain of SAP18, forming a ternary complex that has both RNA- and protein-binding properties. Unexpectedly, our structural analysis identified an Acinus-like motif in Pinin, another EJC-associated splicing factor. We show that Pinin physically interacts with RNPS1 and SAP18, forming an alternative ternary complex, PSAP.

Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.