Structural and functional insights into CWC27/CWC22 heterodimer linking the exon junction complex to spliceosomes.

Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.

CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex.

The exon junction complex (EJC) is a central effector of the fate of mRNAs, linking nuclear processing to mRNA transport, translation and surveillance. However, little is known about its transcriptome-wide targets. We used cross-linking and immunoprecipitation methods coupled to high-throughput sequencing (CLIP-seq) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks that are located mainly in spliced mRNAs. Most expressed exons harbor peaks either in the canonical EJC region, located ~24 nucleotides upstream of exonic junctions, or in other noncanonical regions. Notably, both of these types of peaks are preferentially associated with unstructured and purine-rich sequences containing the motif GAAGA, which is a potential binding site for EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.

Conserved motifs involved in ATP hydrolysis by MalT, a signal transduction ATPase with numerous domains from Escherichia coli.

The signal transduction ATPases with numerous domains (STAND) are sophisticated signaling proteins that are related to AAA+ proteins and control various biological processes, including apoptosis, gene expression, and innate immunity. They function as tightly regulated switches, with the off and on positions corresponding to an ADP-bound, monomeric form and an ATP-bound, multimeric form, respectively. Protein activation is triggered by inducer binding to the sensor domain. ATP hydrolysis by the nucleotide-binding oligomerization domain (NOD) ensures the generation of the ADP-bound form. Here, we use MalT, an Escherichia coli transcription activator, as a model system to identify STAND conserved motifs involved in ATP hydrolysis besides the catalytic acidic residue. Alanine substitution of the conserved polar residue (H131) that is located two residues downstream from the catalytic residue (D129) blocks ATP hydrolysis and traps MalT in an active, ATP-bound, multimeric form. This polar residue is also conserved in AAA+. Based on AAA+ X-ray structures, we proposed that it is responsible for the proper positioning of the catalytic and the sensor I residues for the hydrolytic attack. Alanine substitution of the putative STAND sensor I (R160) abolished MalT activity. Substitutions of R171 impaired both ATP hydrolysis and multimerization, which is consistent with an arginine finger function and provides further evidence that ATP hydrolysis is primarily catalyzed by MalT multimers.

CORSEN, a new software dedicated to microscope-based 3D distance measurements: mRNA-mitochondria distance, from single-cell to population analyses.

Recent improvements in microscopy technology allow detection of single molecules of RNA, but tools for large-scale automatic analyses of particle distributions are lacking. An increasing number of imaging studies emphasize the importance of mRNA localization in the definition of cell territory or the biogenesis of cell compartments. CORSEN is a new tool dedicated to three-dimensional (3D) distance measurements from imaging experiments especially developed to access the minimal distance between RNA molecules and cellular compartment markers. CORSEN includes a 3D segmentation algorithm allowing the extraction and the characterization of the cellular objects to be processed--surface determination, aggregate decomposition--for minimal distance calculations. CORSEN's main contribution lies in exploratory statistical analysis, cell population characterization, and high-throughput assays that are made possible by the implementation of a batch process analysis. We highlighted CORSEN's utility for the study of relative positions of mRNA molecules and mitochondria: CORSEN clearly discriminates mRNA localized to the vicinity of mitochondria from those that are translated on free cytoplasmic polysomes. Moreover, it quantifies the cell-to-cell variations of mRNA localization and emphasizes the necessity for statistical approaches. This method can be extended to assess the evolution of the distance between specific mRNAs and other cellular structures in different cellular contexts. CORSEN was designed for the biologist community with the concern to provide an easy-to-use and highly flexible tool that can be applied for diverse distance quantification issues.

Wheel of Life, Wheel of Death: A Mechanistic Insight into Signaling by STAND Proteins.

The signal transduction ATPases with numerous domains (STAND) represent a newly recognized class of widespread, sophisticated ATPases that are related to the AAA+ proteins and that function as signaling hubs. These proteins control diverse biological processes in bacteria and eukaryotes, including gene expression, apoptosis, and innate immunity responses. They function as tightly regulated switches, with the off and on positions corresponding to a long-lived monomeric, ADP-bound form and a multimeric, ATP-bound form, respectively. Inducer binding to the sensor domain activates the protein by promoting ADP for ATP exchange, probably through removal of an intramolecular inhibitory interaction, whereas ATP hydrolysis turns off the protein. One key component of the switch is a three-domain module carrying the ATPase activity (nucleotide-binding oligomerization domain [NOD]). Analysis of the atomic structures of four crystallized nucleotide-bound NOD modules provides an unprecedented insight into the NOD conformational changes underlying the activation process.

How integration of positive and negative regulatory signals by a STAND signaling protein depends on ATP hydrolysis.

The role of nucleotide hydrolysis in signaling by signal transduction ATPases with numerous domains (STAND) is poorly understood. Here we use MalT, the transcription activator of the Escherichia coli maltose regulon, as a model system to address this question. We have constructed the MalT-D129A variant that binds ATP but does not hydrolyze it and have characterized it in vivo and in vitro. ATP hydrolysis is not essential for transcription activation but is crucial in controlling MalT activity. MalT cycles between an ADP-bound, resting form that is the target of negative effectors and an ATP-bound, active form, which oligomerizes. Conversion to the active form involves nucleotide exchange and depends on maltotriose binding, whereas resetting to the inactive state relies on ATP hydrolysis, which ensues MalT multimerization. Such a controlled binary switch most likely applies to the other STAND NTPases, including Apaf-1 and the human innate immunity proteins NOD2, and CIAS1.