SummaryAberrantly slow translation elicits quality control pathways initiated by the ubiquitin ligase ZNF598. How ZNF598 discriminates physiologic from pathologic translation complexes and ubiquitinates stalled ribosomes selectively is unclear. Here, we find that the minimal unit engaged by ZNF598 is the collided di-ribosome, a molecular species that arises when a trailing ribosome encounters a slower leading ribosome. The collided di-ribosome structure reveals an extensive 40S-40S interface in which the ubiquitination targets of ZNF598 reside. The paucity of 60S interactions allows for different ribosome rotation states, explaining why ZNF598 recognition is indifferent to how the leading ribosome has stalled. The use of ribosome collisions as a proxy for stalling allows the degree of tolerable slowdown to be tuned by the initiation rate on that mRNA; hence, the threshold for triggering quality control is substrate specific. These findings illustrate how higher-order ribosome architecture can be exploited by cellular factors to monitor translation status.

TRIM5α proteins are restriction factors that protect mammalian cells from retroviral infections by binding incoming viral capsids, accelerating their dissociation, and preventing reverse transcription of the viral genome. Individual TRIM5 isoforms can often protect cells against a broad range of retroviruses, as exemplified by rhesus monkey TRIM5α and its variant, TRIM5-21R, which recognize HIV-1 as well as several distantly related retroviruses. Although capsid recognition is not yet fully understood, previous work has shown that the C-terminal SPRY/B30.2 domain of dimeric TRIM5α binds directly to viral capsids, and that higher-order TRIM5α oligomerization appears to contribute to the efficiency of capsid recognition. Here, we report that recombinant TRIM5-21R spontaneously assembled into two-dimensional paracrystalline hexagonal lattices comprising open, six-sided rings. TRIM5-21R assembly did not require the C-terminal SPRY domain, but did require both protein dimerization and a B-box 2 residue (Arg121) previously implicated in TRIM5α restriction and higher-order assembly. Furthermore, TRIM5-21R assembly was promoted by binding to hexagonal arrays of the HIV-1 CA protein that mimic the surface of the viral capsid. We therefore propose that TRIM5α proteins have evolved to restrict a range of different retroviruses by assembling a deformable hexagonal scaffold that positions the capsid-binding domains to match the symmetry and spacing of the capsid surface lattice. Capsid recognition therefore involves a synergistic combination of direct binding interactions, avidity effects, templated assembly, and lattice complementarity.

Tripartite motif (TRIM) proteins make up a large family of coiledcoil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the Nterminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.antiparallel dimer | disulfide crosslinking | X-ray crystallography T ripartite motif (TRIM) proteins make up the largest superfamily of RING E3 ubiquitin ligases, with more than 100 members in the human proteome (1, 2). TRIM proteins function in a variety of cellular pathways, and many regulate innate immunity and/or mediate antiviral responses. Antiviral TRIMs include TRIM25, which regulates the IFN response to RNA viruses (3, 4), and TRIM5α, which senses and inhibits early stages of retroviral replication (5, 6).TRIM proteins share a common N-terminal domain organization, termed the tripartite or RBCC (RING, B-box, coiled-coil) motif, followed by variable C-terminal protein recognition domains ( Fig. 1 A and B). "Linker" segments of unknown structure typically separate both the RING and B-box domains (L1) and the coiledcoil and terminal effector domains (L2). The coiled-coil region mediates oligomerization, and both homooligomeric and heterooligomeric TRIMs have been described (7-13). Furthermore, many TRIM proteins form higher-order assemblies in vitro and form punctate or fibrous structures in cells (14-16). For example, TRIM5α assembly allows the protein to function as a cytosolic patternrecognition receptor that can intercept the incoming capsids of diverse retroviruses, including HIV-1 (6). This results in species-specific "restriction" of viral replication (5), capsid dissociation (5, 6), and induction of innate immune responses (17). Retroviral capsids are recognized through a remarkable mechanism of multivalent pattern recognition. TRIM5α forms a homodimer (10, 11, 18), which can further assemble into a 2D lattice of linked hexagons (18). The hexagonal TRIM5α net matches the symmetry and spacing of the retroviral capsid surface lattice, thereby positioning multiple C-terminal B30.2/SPRY domains to interact with their repeating binding epitopes on the capsid.Struct...

The rhesus monkey intrinsic immunity factor TRIM5␣ rh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5␣ proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5␣ rh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5␣ proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5␣ proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5␣ proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.Susceptibility to retroviral infections influences species survival and has driven the evolution of cellular restriction factors that inhibit retroviral replication. One important antiretroviral intrinsic immune response is mediated by TRIM5␣, which can block early postentry steps in the replication of certain retroviruses in specific primate lineages (3, 37, 55, 58). Under normal restrictive conditions, TRIM5␣ proteins block accumulation of retroviral reverse transcripts (55) and accelerate the rate at which viral capsids dissociate from high-molecularweight complexes into lower-molecular-weight subunits (42,56). The allelic specificity of TRIM5␣ restriction is illustrated by the fact that rhesus macaque TRIM5␣ potently inhibits human immunodeficiency virus type 1 (HIV-1) replication, whereas human TRIM5␣ instead exhibits restriction activity against N-tropic murine leukemia virus but not 29,43,55,67). These differences can be attributed to the differential abilities of TRIM5␣ proteins to interact with retroviral capsids after viral entry (34,42,56).Like other tripartite (TRIM) family members, TRIM5␣ contains RING, B-box, coiled-coil, and B30.2/SPRY domains, and each of t...

Faulty or damaged mRNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation, and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-tRNA conformation sub-optimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt an rRNA-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids.DOI: http://dx.doi.org/10.7554/eLife.16269.001

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo–electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA) degradation via an unknown mechanism. We identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers cotranslational degradation of tubulin mRNAs in response to excess soluble tubulin. Structural analysis revealed that TTC5 binds near the ribosome exit tunnel and engages the amino terminus of nascent tubulins. TTC5 mutants incapable of ribosome or nascent tubulin interaction abolished tubulin autoregulation and showed chromosome segregation defects during mitosis. Our findings show how a subset of mRNAs can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode.