Jérôme Basquin

RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex.

Eukaryotic Sm and Sm‐like proteins associate with RNA to form the core domain of ribonucleoprotein particles involved in pre‐mRNA splicing and other processes. Recently, putative Sm proteins of unknown function have been identified in Archaea. We show by immunoprecipitation experiments that the two Sm proteins present in Archaeoglobus fulgidus (AF‐Sm1 and AF‐Sm2) associate with RNase P RNA in vivo, suggesting a role in tRNA processing. The AF‐Sm1 protein also interacts specifically with oligouridylate in vitro. We have solved the crystal structures of this protein and a complex with RNA. AF‐Sm1 forms a seven‐membered ring, with the RNA interacting inside the central cavity on one face of the doughnut‐shaped complex. The bases are bound via stacking and specific hydrogen bonding contacts in pockets lined by residues highly conserved in archaeal and eukaryotic Sm proteins, while the phosphates remain solvent accessible. A comparison with the structures of human Sm protein dimers reveals closely related monomer folds and intersubunit contacts, indicating that the architecture of the Sm core domain and RNA binding have been conserved during evolution.

Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family.

The eukaryotic exosome is a macromolecular complex essential for RNA processing and decay. It has recently been shown that the RNase activity of the yeast exosome core can be mapped to a single subunit, Rrp44, which processively degrades single-stranded RNAs as well as RNAs containing secondary structures. Here we present the 2.3 A resolution crystal structure of S. cerevisiae Rrp44 in complex with single-stranded RNA. Although Rrp44 has a linear domain organization similar to bacterial RNase II, in three dimensions the domains have a different arrangement. The three domains of the classical nucleic-acid-binding OB fold are positioned on the catalytic domain such that the RNA-binding path observed in RNase II is occluded. Instead, RNA is threaded to the catalytic site via an alternative route suggesting a mechanism for RNA-duplex unwinding. The structure provides a molecular rationale for the observed biochemical properties of the RNase R family of nucleases.

Structural and Biochemical Insights to the Role of the Ccr4- not Complex and Ddx6 ATPase in Microrna Repression.

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45.

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3′ splice site–recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-Å crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.

An asymmetric SMC-kleisin bridge in prokaryotic condensin

Eukaryotic structural maintenance of chromosomes (SMC)–kleisin complexes form large, ring-shaped assemblies that promote accurate chromosome segregation. Their asymmetric structural core comprises SMC heterodimers that associate with both ends of a kleisin subunit. However, prokaryotic condensin Smc–ScpAB is composed of symmetric Smc homodimers associated with the kleisin ScpA in a postulated symmetrical manner. Here, we demonstrate that Smc molecules have two distinct binding sites for ScpA. The N terminus of ScpA binds the Smc coiled coil, whereas the C terminus binds the Smc ATPase domain. We show that in Bacillus subtilis cells, an Smc dimer is bridged by a single ScpAB to generate asymmetric tripartite rings analogous to eukaryotic SMC complexes. We define a molecular mechanism that ensures asymmetric assembly, and we conclude that the basic architecture of SMC–kleisin rings evolved before the emergence of eukaryotes.

Archaeal Sm proteins form heptameric and hexameric complexes: crystal structures of the Sm1 and Sm2 proteins from the hyperthermophile Archaeoglobus fulgidus.

Proteins of largely unknown function related to the Sm proteins present in the core domain of eukaryotic small nuclear ribonucleoprotein particles have recently been detected in Archaea. In contrast to eukaryotes, Archaea contain maximally two distinct Sm-related proteins belonging to different subfamilies, we refer to as Sm1 and Sm2. Here we report the crystal structures of the Sm1- and Sm2-type proteins from the hyperthermophilic euryarchaeon Archaeoglobus fulgidus (AF-Sm1 and AF-Sm2) at a resolution of 2.5 and 1.95 A, respectively. While the AF-Sm1 protein forms a heptameric ring structure similar to that found in other archaeal Sm1-type proteins, the AF-Sm2 protein unexpectedly forms a homo-hexamer in the crystals, and, as is evident from the mass spectrometric analysis, also in solution. Both proteins have essentially the same monomer fold and inter-subunit beta-sheet hydrogen bonding giving rise to a similar overall architecture of the doughnut-shaped six and seven-membered rings. In addition, a conserved uracil-binding pocket identified previously in an AF-Sm1/RNA complex, suggests a common RNA-binding mode for the AF-Sm1 and AF-Sm2 proteins, in line with solution studies showing preferential binding to U-rich oligonucleotides for both proteins. Clear differences are however seen in the charge distribution within the two structures. The rough faces of the rings, i.e. the faces not containing the base binding pockets, have opposite charges in the two structures, being predominantly positive in AF-Sm1 and negative in AF-Sm2. Differences in the ionic interactions between subunits provide an explanation for the distinctly different oligomerisation behaviour of the AF-Sm1 and AF-Sm2 proteins and of Sm1- and Sm2-type proteins in general, as well as the stability of their complexes. Implications for the functions of archaeal Sm proteins are being discussed.

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.

Crystallization of biological macromolecules using agarose gel

Gellified media prevent convection and crystal sedimentation, and provide an attractive growth environment for optimising biological crystals. Agarose gels are particularly easy to use and they are compatible with most of the common crystallization methods. They also offer new possibilities like counter-diffusion techniques. This paper gives a brief overview of their general properties and presents an application of a counter-diffusion setup combining agarose gel and capillaries to the crystallization of proteins and protein / nucleic acid complexes.

Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes.

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 Å, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors.

Structural and biochemical characterization of the yeast exosome component Rrp40

The exosome is a protein complex that is important in both degradation and 3′‐processing of eukaryotic RNAs. We present the crystal structure of the Rrp40 exosome subunit from Saccharomyces cerevisiae at a resolution of 2.2 Å. The structure comprises an S1 domain and an unusual KH (K homology) domain. Close packing of the S1 and KH domains is stabilized by a GxNG sequence, which is uniquely conserved in exosome KH domains. Nuclear magnetic resonance data reveal the presence of a manganese‐binding site at the interface of the two domains. Isothermal titration calorimetry shows that Rrp40 and archaeal Rrp4 alone have very low intrinsic affinity for RNA. The affinity of an archaeal core exosome for RNA is significantly increased in the presence of the S1–KH subunit Rrp4, indicating that multiple subunits might contribute to cooperative binding of RNA substrates by the exosome.