Nature Structural & Molecular Biology

Nick translation during Okazaki fragment maturation requires iterative coordination of DNA polymerase delta (Pol {delta}), which displaces the downstream primer, and flap endonuclease FEN1, which cleaves the resulting flap, on the sliding clamp PCNA. The structural basis of this coordination is unknown. We present cryo-EM structures of human Pol {delta}-PCNA, Pol {delta}-PCNA-FEN1 and FEN1-PCNA on flap DNA, capturing four states of the nick translation cycle. Pol {delta} strand displacement emerges from structural elements intrinsic to the B-family fold rather than dedicated separation machinery, with a conserved palm loop acting as separation wedge and PCNA engagement required for melting of the downstream duplex. In the Pol {delta}-PCNA-FEN1 toolbelt, FEN1 is pre-positioned opposite Pol {delta} on PCNA to receive the substrate. Nucleotide removal triggers DNA handoff while both enzymes remain clamp-bound, followed by Pol {delta} dissociation. A post-handoff structure reveals stable DNA retention by FEN1-PCNA after flap cleavage, explaining the slow nick translation kinetics and the obligate role of Ligase 1 in sealing.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/741260v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@fd2b90org.highwire.dtl.DTLVardef@1371d4borg.highwire.dtl.DTLVardef@164661forg.highwire.dtl.DTLVardef@6cae26_HPS_FORMAT_FIGEXP M_FIG C_FIG Key findingsO_LICryo-EM reveals SDE2 as a novel factor stabilizing the spliceosome active center C_LIO_LISDE2 {Delta}N structures visualize a stalled C* state with impaired docking-factor engagement. C_LIO_LISDE2, Prp18, and FAM32A read a cis-code to stabilize weaker proximal 3'-ss. C_LIO_LISDE2 {Delta}N destabilizes 3'-ss docking and rescues BRCA1 and CFTR mis-splicing in vivo. C_LI Accurate alternative splicing requires discrimination between adjacent 3' splice sites (3'-ss) during catalysis and is disrupted by pathogenic AG-gain mutations that create competing 3'-ss. Here, we present cryo-EM structures of human spliceosomes assembled on native-sequence pre-mRNAs, revealing how the catalytic core controls alternative 3'-ss selection. SDE2 is a previously unrecognized active-center component that promotes a docking-competent spliceosome conformation. Machine learning, in vivo transcriptomics, and in vitro biochemistry show how SDE2 cooperates with FAM32A and Prp18 to act as readers of a cis-regulatory code that governs 3'-ss selection during catalysis. These factors promote weaker, proximal site use by counteracting an intrinsic distal bias generated by active-site interactions with the distal-site -4 nucleotide. Structural or genetic perturbation of these exon-ligation factors destabilizes proximal 3'-ss docking and restores canonical splicing in disease-relevant CFTR and BRCA1 AG-gain alleles. Our work establishes the spliceosome active center as a tunable regulatory hub for alternative splicing.

The SLC6 family is a major target for neuropsychiatric therapeutics. Human B0AT2 (SLC6A15) regulates cerebral amino acid homeostasis and glutamatergic transmission and has been linked to major depressive disorder, yet its transport and inhibition mechanisms remain unclear. Here we report cryo-EM structures of human B0AT2 in the apo state and in complex with three substrates (proline, leucine, and methionine) and two inhibitors (loratadine and tiagabine), capturing outward-open, early substrate-bound intermediate, outward-occluded, and inward-open conformations along the transport cycle. These structures reveal a local conformational tuning at the canonical substrate-binding pocket (S1), in which rearrangement of Phe308 remodels the pocket geometry to tune substrate accommodation and selectivity. Loratadine stabilizes an outward-occluded state via allosteric inhibition at the extracellular S2 pocket, whereas tiagabine stabilizes the inward-open state through cooperative multi-site inhibition involving the S1 site and two previously unrecognized intracellular cavities (S3 and S4). Together with functional assays and mutagenesis, these data define the molecular basis of B0AT2 substrate selectivity and state-dependent inhibition. Notably, the two newly identified intracellular cavities are broadly conserved within the SLC6 family, reflecting a common intracellular vestibular architecture and enabling the rational design of conformation-selective modulators for neuropsychiatric disorders.

Cellular processes are compartmentalized within immiscible heterotypic condensates, yet the functional consequences of losing their physical segregation remain unclear. Here, we show that genetic inactivation of the RNA chaperone SMN forces the aberrant intermixing of the two most prominent nuclear condensates: nucleolus and Cajal Body (CB). Upon SMN depletion, CB components invade the nucleolus and undergo reduced mobility and solubility consistent with a liquid-to-gel-like hardening transition. The CB-scaffold coilin aberrantly enriches at the nucleolar FC/DFC boundary and occupies rDNA chromatin, thereby locally suppressing rRNA production. Concurrently, this sequestration globally impairs coilin targeting to snRNA/snoRNA loci and limits telomerase access to telomeres, reducing telomeric synthesis. Crucially, genetic coilin depletion alone alleviates this mistargeting and rescues these functional impairments across condensates. Our findings reveal an inter-condensate rheostat model in which the loss of CB-nucleolar immiscibility is directly sensed, communicated, and executed by CB remnants, thereby proportionally coupling the functional outputs of otherwise distinct RNPs essential for splicing, translation, and genomic integrity.

PIWI proteins are members of the Argonaute family and together with piRNAs protect metazoan germlines from transposons. PIWI proteins adopt a bi-lobed architecture with a central RNA-binding channel. HSP90 function has been linked to piRNA biogenesis, but the precise molecular mechanism is unresolved. Using the mammalian embryonic piRNA pathway as a model system, we find compelling evidence for the existence of PIWIL2- (MILI-) and PIWIL4- (MIWI2-) HSP90 complexes in foetal testis. We purify apo-PIWIL4-HSP90 from cells and determine its structure by cryo-electron microscopy. Distinct from piRNA-bound PIWI, apo-PIWIL4 adopts a unique and open conformation. The HSP90 dimer binds and unfolds PIWIs linker 1 domain. PIWIL4s N domain and the RNA-binding PAZ-MID-PIWI module are placed on opposite sides of the HSP90 dimers lumen. We further demonstrate that PIWI-HSP90 complexes, the open apo-PIWI conformation, and the HSP90 lumen-binding peptide are conserved features of PIWI proteins.

Alternative pre-mRNA splicing generates extensive transcript diversity, yet the regulatory code that determines how splicing decisions are encoded across the transcriptome remains poorly defined. Splicing outcomes are controlled by combinatorial RNA-binding protein (RBP) interactions and positional context, but how these features are integrated at the transcriptome scale remains unclear. CLIP-based approaches have mapped RBP binding, but directly comparable endogenous maps across multiple RBPs are lacking, limiting inference of global regulatory principles. Here we introduce SCALE-CLIP, an endogenous CLIP framework that integrates CRISPR-Cas9-mediated epitope tagging with long-read-guided read attribution to generate directly comparable RBP binding maps across splicing-regulatory factors. Applied to 23 RBPs, SCALE-CLIP expanded endogenous RBP coverage and, across benchmarked shared factors, increased peak recovery by a median of 12.2-fold relative to ENCODE eCLIP while preserving specificity and reproducibility. We define a transcriptome-wide positional and combinatorial code for alternative splicing, in which binding position is a primary determinant of regulatory outcome: SRSF binding within alternative exons promotes inclusion, whereas binding on flanking exons drives exon skipping. Higher-order SRSF occupancy further tunes this code, buffering exon inclusion when centered on alternative exons but reinforcing repression when distributed across flanking exons. We also show that m6A provides an epitranscriptomic layer that locally enhances SRSF binding and is associated with increased exon inclusion. Together, these results establish a multi-layered RNA-binding logic in which binding position, combinatorial RBP architecture and RNA modification jointly shape splicing outcomes, providing a framework for rational interpretation and modulation of alternative splicing.

The ubiquitin-proteasome system represents the main pathway for targeted protein degradation in eukaryotic cells. The majority of substrates is recruited for degradation through ubiquitin modifications, and the underlying principles are well established. However, the requirements for ubiquitin-independent substrates are still poorly understood. Here, we reveal the mechanisms for the antizyme-mediated degradation of the yeast ornithine decarboxylase (yODC), the first reported ubiquitin-independent substrate of the 26S proteasome. Using biochemical studies and cryo-EM structure determination, we show how antizyme binding makes the yODC monomer prone for degradation by exposing an interface that is normally buried in the catalytically active ODC dimer. Together with a surface on antizyme, yODC forms a two-part interface that binds the N-terminal coiled coil of two ATPase subunits, Rpt4 and Rpt5, for delivery to the 26S proteasome motor. This positions the N-terminal unstructured region of yODC for insertion into the ATPase channel to initiate degradation, which we found does not depend on a specific sequence. Interestingly, binding of the globular yODC/antizyme complex to the Rpt4/Rpt5 coiled coil allosterically stabilizes a proteasome conformation that facilitates substrate engagement by the ATPase motor and may represent a primed pre-initiation state with a general role in ubiquitin-dependent and -independent degradation.

How the earliest structure within the unfolded state is formed during biosynthesis on the ribosome and whether it has any consequences for downstream folding remain open questions. Here, we combine 15N paramagnetic relaxation enhancement NMR with all-atom molecular dynamics simulations to characterise the unfolded state of a folding-competent immunoglobulin-like domain on the ribosome at the cusp of folding initiation. We identify three structurally distinct sub-ensembles that differ in compaction and ribosome interactions. Non-native contacts, together with ribosome interactions, likely delay folding, yet their persistence alongside early native-like contacts within a sparsely populated compact sub-ensemble suggests they may also facilitate the formation of a co-translational folding nucleus, whose contacts overlap with those of the downstream intermediates. From these findings we infer a mechanistic model of de novo folding initiation during biosynthesis and, by linking the folding nucleus to downstream partially structured intermediates and the native state, provide a complete atomistic description of a co-translational folding pathway.

Picornaviruses are one of the leading agents of animal and human infectious disease with at least 8 billion infections a year and cause a range of symptoms including respiratory failure and acute flaccid myelitis1. The most conserved nonstructural protein in picornaviruses is 2C2, a member of the AAA+ family of ATPases that binds RNA, and a broad spectrum antiviral target3-5. Despite its crucial role in the viral life cycle and as a clinical target, no structure of 2C bound to RNA has been structurally determined. Here we present the first structure of 2C as a hexamer bound to single stranded RNA in its central pore; a novel AAA+ protein:substrate interaction. Using the 2C:RNA holoenzyme complex structure, we characterize the mode that this AAA+ protein employs to specifically bind single stranded RNA, and demonstrate that mutations to key residues inhibit both RNA binding and viral replication in Apthovirus and Enterovirus systems, and show that the core residues responsible for binding are broadly conserved in viruses beyond Picornaviridae. Finally, we reveal that the 2C:RNA holoenzyme complex is conformationally more similar to a protein translocase adapted to bind RNA rather than other viral DNA binding SF3 helicases, underscoring how the AAA+ core module can be adapted for a variety of biochemical substrates.

G protein-coupled receptors (GPCRs) signal across a continuum of activation states, yet how ligands encode distinct signaling efficacies remains poorly understood. Here, we define the molecular mechanism underlying graded activation of the dopamine D1 receptor (D1R), a major therapeutic target for neuropsychiatric disorders. Functional analyses reveal that two widely used pharmacological tools, LE300 and SCH23390, possess intrinsic efficacy as an inverse agonist and a weak partial agonist, respectively, rather than the efficacy-silent neutral antagonists. Structural, molecular dynamics and mutagenesis analyses capture previously unrecognized inactive and intermediate receptor activation states that bridge known active conformations, and reveal that ligand efficacy is encoded through the progressive engagement of a conserved activation pathway centered on the W6.48 toggle switch. Guided by this mechanism, a single chemical modification markedly increases the agonist efficacy of SCH23390. Comparison with dopamine D2 receptor structures further reveals a conserved mechanism of inverse agonism despite distinct subtype-specific recognition. Together, these findings establish a structural framework for graded agonism at D1R and provide general principles for the rational design of efficacy-tuned therapeutics at dopamine receptors and related GPCRs.

Translation initiation requires messenger RNAs (mRNAs) to be recognized and loaded into ribosomes through a process catalyzed by the heterotrimeric eukaryotic initiation factor eIF4F. During this process, eIF4F engages the 7-methylguanosine cap at the 5 end of the mRNA and promotes productive engagement with the ribosomal pre-initiation complex (PIC) to facilitate PIC loading onto the mRNA. Although eIF4F is central to translation initiation and its regulation, the molecular mechanism by which eIF4F stimulates PIC loading, and the mechanistic role of the essential ATP hydrolysis step catalyzed by eIF4F, have remained unresolved. Here, we use single-molecule fluorescence microscopy to directly visualize the dynamics of eIF4F during cap recognition and PIC engagement. We show that ATP binding, but not ATP hydrolysis, promotes productive assembly of eIF4F on mRNA and enables dynamic redistribution of eIF4F along the transcript. In contrast, ATP hydrolysis is specifically required for recycling of cap-stalled eIF4F during productive PIC engagement. Furthermore, we identify eIF3 and eIF4B as the minimal PIC-associated factors required to stimulate ATP-hydrolysis-dependent recycling of eIF4F during PIC loading. Together, our results support a model in which productive PIC engagement stimulates ATP-hydrolysis-dependent recycling of eIF4F, thereby coupling eIF4F recycling to PIC loading during translation initiation. This mechanism provides a framework for understanding how mRNA topology, RNA-binding proteins, and the availability of initiation factors can control translational efficiency.

Human endogenous retroviruses (hERVs) are remnants of ancestral retroviral infections that have shaped the human genome through their capacity to mobilize and retrotranspose. Among these, hERV-K (HML-2) remains the most recently active family and its dysregulation is strongly associated with diverse cancers and neurodegenerative pathologies, yet the structural basis of its integration remains poorly understood. Here, we combine activity assays with high-resolution cryo-electron microscopy to resolve the hERV-K integration machinery in three distinct states: asymmetric target-DNA engagement, strand transfer, and pharmacological inhibition. Our structures reveal a compact architecture defined by a unique organization of the outer integrase domains, which distinguishes hERV-K from other known retroviral intasomes. Biochemical validation confirms the catalytic competence of this compact tetrameric assembly, which relies on specialized polar motifs to optimize synaptic stability while retaining sensitivity to competitive antagonism by strand transfer inhibitors. Notably, beyond canonical restriction by Raltegravir, we discovered that the drug binding stabilizes an unanticipated, "closed" conformation not observed in previously characterized intasomes. Together, these findings elucidate the molecular mechanism of endogenous retroviral integration and provide a structural framework for rational therapeutic targeting of hERV-K-driven diseases.

Replication risk sequences (RRS) are recently discovered genomic structural elements that trigger post-replication gap formation during replisome passage. In E. coli, the two RRS elements are nearly perfect 222-bp G-quadruplex-containing repeats that flank the terminal domain and are highly conserved in both sequence and genomic position across enterobacteria. We report here the first direct visualisation of RRS function in vitro using single-molecule methods. When the G4 strand of the RRS element is positioned on the lagging-strand template, gaps are formed essentially every time a replisome encounters it. An increase in ssDNA in the synthesised DNA is readily seen using ssGAP-seq methods. When the G-quadruplex strand of the RRS is positioned on the leading-strand template, gaps are formed, albeit at lower frequency. However, the continued DNA synthesis in a rolling-circle assay indicates that the gaps are still formed on the lagging strand, indicating that the RRS complementary strand has a significant but reduced capacity to form a structure that triggers lesion skipping. The results document the potency of the RRS as a trigger for gap formation, suggesting a possible function for at least some eukaryotic G-quadruplexes.

From S phase until anaphase, sister chromatids remain physically linked to ensure accurate chromosome segregation. This linkage, called sister chromatid cohesion, must withstand the pulling forces generated by the mitotic spindle and is thought to require entrapment of sister DNAs inside the ring-shaped cohesin complex. DNA catenations that arise naturally during replication could also tether sister chromatids, but whether they contribute to cohesion has remained unresolved. Here, using budding yeast, we show that cohesin-mediated sister DNA entrapment alone cannot withstand spindle-generated pulling forces and that robust sister chromatid cohesion requires DNA catenation. Selective removal of DNA catenations causes catastrophic cohesion loss, delays chromosome biorientation and increases chromosome mis-segregation, even when cohesin rings remain intact. Importantly, DNA catenation similarly underpins force-resistant cohesion in metazoan chromosomes. Our findings fundamentally redefine the physical basis of sister chromatid cohesion by establishing DNA catenation as an evolutionarily conserved component of the force-resistant linkage between sister chromatids that enables accurate chromosome segregation.

Ribosome composition can vary through differences in associated proteins, post-transcriptional and post-translational modifications. Such heterogeneity enables ribosomes to respond to environmental1 or pathological2,3 conditions, and modulate localized translation4. A long-standing observation has also been the differential expression of variant ribosomal RNA (rRNA) alleles across developmental5-7 or cellular states8-14. Yet how exchanging the catalytic ribosome core could regulate translational outcomes remains unknown. Here, we report the functional characterization of a genomically-encoded, divergent rRNA that serves as a dominant-negative repressor of translation during host-to-vector transmission in the human malaria parasite. This allele only encodes for large subunit rRNAs, lacks ITS2 splicing, yet retains conserved rRNA modification and folding patterns alongside vast expansion segments. The resulting large subunit engages mRNA at translation start sites but appears to elongate inefficiently, likely due to divergences in the peptidyl transferase center obstructing the exit tunnel. Through its precisely timed transcription immediately after transmission, this rRNA represses mRNAs that were highly translated in the human, facilitating the transition of the translational program for mosquito-stage development. Our data identify a repressive ribosome population whose antagonistic function is encoded by an independently evolved, variant rRNA allele, defining the conceptual foundation for an additional layer of inherent translational regulation.

The tuberous sclerosis complex (TSC) protein complex (TSCC) acts as the GTPase activating protein (GAP) for the small GTPase Rheb, to limit mTORC1 (mechanistic target of rapamycin complex 1) activity and cellular growth. We report the structure of the catalytic transition state complex of TSCC and Rheb. Assembly of two TSC2 subunits containing "asparagine-thumb" GAP domains with two accessory TSC1 subunits is required for function in cells. Catalysis requires the "asparagine-thumb" residue of TSC2 and conserved residues in Rheb that bind TSC2 at multiple interaction sites. Surprisingly, only one TSC2 GAP domain is catalytically competent and interacts with Rheb. This is realized by asymmetric binding of TSC1, which enables activating structural changes in one of the TSC2 subunits and locks the second TSC2 copy in an inactive conformation. We identify TSC2 variants that affect conformational coupling within TSCC and binding to Rheb. The structure thus explains the catalytic mechanism of TSCC and reveals an allosteric role of TSC1 in this process.

Bacterial translation initiation is a highly regulated process essential for accurate start codon selection and the assembly of an elongation-competent ribosome. Two initiation factors, 1 (IF1) and 3 (IF3), contribute to quality control for formation of 30S preinitiation complex (30S PIC), while the GTPase IF2 facilitates stable initiator tRNA binding and promotes subunit association. However, the molecular mechanism of IF1 action and the regulatory role of IF2-mediated GTP hydrolysis and inorganic phosphate (Pi) release remain poorly understood. Using ensemble cryo-EM integrated with fast-kinetics, we delineate the translation initiation pathway involving IF1 and IF2. We show that IF1 transiently associates with the 30S subunit and interferes with the formation of multiple inter-subunit bridges. IF2 promotes subunit association by stabilizing the 30S PIC through interactions mediated by its N-terminal domains. IF1 departure happens after or concomitant with GTP hydrolysis, following which the inter-subunit bridges establish. Then Pi release triggers remodeling of IF2 followed by its departure from the 70S initiation complex. These findings reveal how the coordinated interplay of IF1 and IF2 with the ribosome ensures translational fidelity and plays crucial role for formation of elongation-competent 70S.

The protein-tyrosine phosphatase SHP2 (PTPN11) regulates growth factor- and cytokine-induced RAS/ERK MAP kinase (MAPK) pathway activation, and aberrant SHP2 function causes developmental disorders and cancer1-5. It is widely believed that the catalytic activity of SHP2 is essential for pathway activation1,4,6-8. This view has shaped our interpretation of how germline PTPN11 mutations cause Noonan Syndrome (NS) and NS with Multiple Lentigines (NS-ML)2,9 and how somatic mutations contribute to myeloproliferative neoplasms and solid tumors1. Here we identify a previously undetected, protein-tyrosine phosphatase (PTP) activity-independent mechanism that revises our understanding of how SHP2 promotes RAS/ERK activation. We find that certain mutations of the nucleophilic cysteine that abolish catalytic activity still promote RAS/ERK pathway activation in normal and neoplastic mammalian cells, zebrafish embryos, and mice. Structural studies show that the SHP2 PTP domain binds directly to the Son of Sevenless 1 (SOS1) Dbl homology (DH) domain. Proximity labeling and super-resolution microscopy demonstrate that SHP2/SOS1 interaction occurs in cells and facilitates SOS1 translocation to the plasma membrane to form clusters. Our results overturn decades of dogma on SHP2 regulation of the RAS/ERK pathway and provide new insights into the mechanism of action of disease-associated PTPN11 mutations.

Preferential orientation at the air-water interface (AWI) remains a persistent challenge in cryo-electron microscopy, often requiring extensive optimization or specialized grid preparation strategies. Here, we show that late embryogenesis abundant (LEA) proteins, previously shown to protect fragile samples from AWI-induced damage, can be leveraged to resolve sample orientation bias. The addition of LEA proteins can yield either new orientation distributions or more isotropic ones in samples suffering from orientation biases. These findings establish LEA proteins as a practical class of cryo-EM additives that can reduce AWI-induced damage and improve particle-orientation distributions in single-particle analysis.

Human APOBEC3B (A3B) restricts virus infections by catalyzing the deamination of cytosines to uracils in single-stranded DNA. A3B also contributes to mutagenesis and genome instability in cancer cells, driving tumor evolution and detrimental outcomes including therapy resistance and metastasis. A3B comprises tandem globular deaminase domains, with a multifunctional amino-terminal domain (NTD) and a catalytically active carboxy-terminal domain (CTD). Although individual domain structures have been studied, the structure of full-length A3B has remained elusive. Here, we report the cryoEM structure of wildtype A3B in complex with the natural antagonist BORF2 (the large subunit of the Epstein-Barr virus ribonucleotide reductase). The two domains of A3B bridge a novel BORF2 dimer interface, showing a unique domain positioning that distinguishes A3B from the related dual-domain retrovirus restriction factor APOBEC3G (A3G). Mutational analyses suggest that the unique NTD-CTD interaction regulates A3B deaminase activity. The BORF2 dimerization interface is stabilized by primary interactions with A3B-CTD and secondary contacts with A3B-NTD, as well as by A3B CTD-CTD dimerization. This matrix of interactions supports a molecular mechanism for A3B neutralization in which BORF2 binding leads to deaminase sequestration in large aggregates. The full-length wildtype A3B structure also provides a platform for future anti-viral and anti-cancer drug development efforts.