Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Dynamic association of human Ebp1 with the ribosome.

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.

A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors.

mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules, we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through their well-characterized interacting proteins results in quantitative differences in RNA mobility. This methodology provides a framework for identifying RBPs that mediate associations with motors.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

The structural basis for RNA selectivity by the IMP family of RNA-binding proteins.

The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

Architecture of the ubiquitylation module of the yeast Ccr4-Not complex.

The Ccr4-Not complex regulates eukaryotic gene expression at multiple levels, including mRNA turnover, translational repression, and transcription. We have studied the ubiquitylation module of the yeast Ccr4-Not complex and addressed how E3 ligase binds cognate E2 and how it is tethered to the complex. The 2.8-Å resolution crystal structure of the N-terminal RING domain of Not4 in complex with Ubc4 shows the detailed interactions of this E3-E2 complex. The 3.6-Å resolution crystal structure of the C-terminal domain of the yeast Not4 in complex with the C-terminal domain of Not1 reveals how a largely extended region at the C-terminus of Not4 wraps around a HEAT-repeat region of Not1. This C-terminal region of Not4 is only partly conserved in metazoans, rationalizing its weaker Not1-binding properties. The structural and biochemical data show how Not1 can incorporate both the ubiquitylation module and the Not2-Not3/5 module concomitantly in the Ccr4-Not complex.

Structure and RNA-binding properties of the Not1-Not2-Not5 module of the yeast Ccr4-Not complex.

The Ccr4-Not complex is involved in several aspects of gene expression, including mRNA decay, translational repression and transcription. We determined the 2.8-Å-resolution crystal structure of a 120-kDa core complex of the Saccharomyces cerevisiae Not module comprising the C-terminal arm of Not1, Not2 and Not5. Not1 is a HEAT-repeat scaffold. Not2 and Not5 have extended regions that wrap around Not1 and around their globular domains, the Not boxes. The Not boxes resemble Sm folds and interact with each other with a noncanonical dimerization surface. Disruption of the interactions within the ternary complex has severe effects on growth in vivo. The ternary complex forms a composite surface that binds poly(U) RNA in vitro, with a site at the Not5 Not box. The results suggest that the Not module forms a versatile platform for macromolecular interactions.

Extended C-terminus and length of the linker connecting the G-domains are species-specific variations in the EngA family of GTPases.

EngA is an essential protein involved in ribosome biogenesis. It is an unique GTPase, possessing two consecutive G-domains. Using sequence and phylogenetic analysis, we found two intriguing variants among EngA homologues - one with a shorter linker joining the G-domains and another with a longer linker, which additionally possesses an extended C-terminus. Interestingly, while the former variant is mainly restricted to firmicutes, the latter is found in nonfirmicutes. Chimeric proteins with interchanged linkers and extensions were generated to gauge the importance of these elements. Ribosome interaction experiments employing the chimeric proteins suggest that a precise combination of the linker and C-terminal extension are important features regulating EngA ribosome interactions in a variant-specific manner.

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.