Nature Structural & Molecular Biology

In Saccharomyces cerevisiae, prolonged cellular stress induces some introns to accumulate post-splicing as stable, linear, spliceosome-protected RNAs1. These stable introns are defined by having short distances from their branchpoint (BP) sequences to their 3'-splice sites (3'SSs). Stable introns sequester splicing components, thereby reducing splicing activity and affecting cell growth in the stressed conditions. The mechanism by which these normally ephemeral products of pre-mRNA splicing persist cannot be explained by the current understanding of the splicing pathway, which derives primarily from studies of unstressed cells and their extracts2,3. Here, we determined the cryo-electron microscopy (cryo-EM) structure of a stable-intron complex purified from saturated-culture conditions. This structure and experimental follow-up show that a Bact-like spliceosome protects stable introns from degradation, and that the short BP-3'SS distances of stable introns render this conformation of the spliceosome resistant to remodelling by helicases. Spliceosomes can also assemble onto artificial introns that have the same sequences as authentic stable introns but do not rely on splicing for their biogenesis, which demonstrates that spliceosomes arrive at this Bact-like conformation by reassembling onto linear introns after their excision from pre-mRNAs. This reassembly activity is maintained in both stressed and unstressed cells. Thus, most yeast introns compete with pre-mRNAs for access to the splicing machinery, and budding yeast has co-opted this activity to adapt to environmental insults.

Linker histones are essential for chromatin compaction, yet how they contribute to higher-order fiber assembly remains poorly understood. Here, we determined cryo-electron microscopy structures of Arabidopsis dodeca-nucleosome fibers containing distinct H2A/H3 variants and linker histone H1.3, revealing a noncanonical binding mode that a laterally positioned H1.3 connects the acidic patch of one nucleosome and the DNA of the neighboring nucleosome, thereby scaffolding dinucleosomes into two-start chromatin fibers. This noncanonical binding mode is structurally conserved when H1.3 is replaced by Gallus gallus H5. Furthermore, incorporation of H2A.W and H3.3 further induces back-to-back fiber dimerization. Cryo-electron tomography and in vivo cross-linking mass spectrometry analyses support the physiological relevance of H1 lateral engagement. Our findings establish that linker histones act as active architectural scaffolds in higher-order chromatin organization.

Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is elusive. Using in vivo spliceosome capture and cryogenic electron microscopy, we determined structures of precatalytic spliceosomes arrested by RBM5 immediately after U2 snRNP branchpoint recognition. Despite intron diversity, the U2-pre-mRNA duplex, branchpoint adenine, and downstream polypyrimidine tract are well-resolved. RBM5 binds the outer SF3B1 HEAT surface and performs dual functions: First, its helix-loop-helix motif and upstream zinc-finger domain sterically block tri-snRNP and Prp8 docking and prevent progression to pre-B and Bact complexes; Second, its G-patch activates DHX15 and places this DExH-box helicase on the pre-mRNA as it exits SF3B1, poised for branch helix unwinding. DHX15 binding to SF3B1 is facilitated by U2SURP/SR140, which engages SF3B1 near RBM5s helix-loop-helix. Functional assays confirm that disruption of the RBM5 interfaces with either DHX15 or SF3B1 inhibit exon repression. Mutations at these regulatory interfaces are common in cancer genomes and predicted to disrupt its regulation of apoptotic isoforms. Thus, RBM5 acts as a dual-action spliceosome gatekeeper that couples helicase activation with physical stalling to enforce tumour-suppressive alternative splicing programmes.

The cyclin-dependent kinase CDK11 functions in transcription, mitotic progression, and mRNA splicing. Specifically, spliceosome activation during the B to Bact transition depends on phosphorylation of the U2 snRNP component SF3B1 by the CDK11-cyclin L-SAP30BP complex. Here, we present the structure of this spliceosome-activating CDK-cyclin complex, determined by cryogenic electron microscopy at 2.3 [A] resolution. Our structure and biochemical experiments show that SAP30BP forms extensive interactions with cyclin L2, thereby stabilising it, and forms critical interactions with the C-terminal kinase lobe of CDK11 that promote complex assembly. Destabilisation of cyclin L2 in the absence of SAP30BP suggests that these principles are applicable to all CDK11-cyclin L complexes. Furthermore, we identify a pseudo-substrate sequence near the CDK11 C-terminus and provide evidence for a role of this segment in CDK11 auto-regulation. Finally, the structure of the CDK11-cyclin L2-SAP30BP complex bound to the clinical high-affinity CDK11 inhibitor OTS964 and a comparison to OTS964-bound off-target complexes provide insight into the mechanism of OTS964 selectivity and specificity.

In most cells, Malat1 long noncoding RNA localizes to the nucleus where it affects splicing and chromatin function. In neurons Malat1 is exported to the cytoplasm where it is translated to generate the M1 micropeptide. Here we characterize an internal ribosome entry site (IRES) required for Malat1 translation. Although preceded by a long Malat1 5 RNA segment this element induces translation at the M1 AUG. In vivo chemical probing and structural modeling identified a 135 nt RNA secondary structure consisting of three stem loops that is sufficient for IRES activity. Using this minimal element for affinity purification from cell extracts, the IRES RNA selectively binds ribosomal subunits and translation factors. Depletion of the binding proteins Rack1 and hnRNP A2/B1 inhibits downstream IRES-dependent translation without affecting translation of an upstream ORF. Our study identifies an unexpected functional unit hidden within a widely studied long noncoding RNA.

In Caenorhabditis elegans, the condensin IDC complex represses transcription from both X chromosomes in hermaphrodites to achieve dosage compensation. How condensin IDC is specifically recruited to the X chromosomes in coordination with sex determination and dosage compensation (SDC) proteins and how it modulates gene expression have, however, remained unresolved. Here, we identify SDC-3 as the key adaptor that directly binds the elbow coiled-coil domain of the condensin IDC-specific SMC subunit DPY-27. Using cryo-electron microscopy, we determine the structure of the SDC-3 adaptor domain bound to an auto-inhibited condensin IDC holoenzyme. Disrupting this interaction compromises dosage compensation and diminishes condensin IDC enrichment on the X chromosomes. Upon overcoming auto-inhibition, condensin IDC exhibits robust DNA loop-extrusion activity comparable to that of canonical condensin. We propose that SDC-3-anchored condensin IDC exploits loop-extrusion to reorganize X-chromosome chromatin and mediate chromosome-wide transcriptional repression.

Nucleosomes with their associated modifications underlie genome organisation and regulation. Replication and transcription require nucleosome disassembly to access the DNA template. How this is orchestrated without jeopardizing chromatin function remains unknown. Here, we reveal a general, global requirement of the histone chaperone FACT in mammalian replication, transcription and chromatin maintenance. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome-wide and chromatin structure in their wake collapses with reduced nucleosome occupancy, irregular spacing and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. This establishes a unifying mechanistic basis for mammalian chromatin replication and transcription, with FACT mediating nucleosome disruption and re-assembly and thereby guarding against spurious chromatin aggregation. Nucleosome organization can therefore dynamically regulate genome architecture.

Cfr methylates C8 of adenosine 2503 (A2503) in 23S ribosomal RNA (rRNA) and will also methylate C2 of A2503 after methylating C8. C8 methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size ([~]36 kDa), we exploit a transient protein-RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 [A] resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome. One Sentence SummaryCryo-EM structure of Cfr, a radical S-adenosylmethionine methylase that confers antibiotic resistance

Upon DNA damage, chromatin remodeling is rapidly initiated to promote chromatin accessibility, thereby facilitating the recruitment and assembly of repair factors. Although this enhanced accessibility has been linked to poly(ADP-ribose) polymerase (PARP) activity, the mechanism by which cells overcome the nucleosome barrier remains unclear. Using our designer chromatin system, we uncovered a previously uncharacterized activity of PARP1, whereby it directly and asymmetrically evicts histone dimers proximal to DNA strand breaks from nucleosomes to generate oriented hexasomes. In the presence of HPF1, PARP1 generates stable PARylated hexasomes, an open chromatin intermediate that can serve as a bifunctional hub for recruitment of DNA- and PAR-dependent factors. Using cellular assays, we demonstrated that PARP activity is both required and sufficient to drive chromatin accessibility and the recruitment of repair factors, with direct involvement of subnucleosomal species. Unexpectedly, we identified the C-terminal tail of histone H2A, a motif harboring recurrent cancer-associated mutations, as a critical determinant of efficient PARP1-mediated nucleosome disassembly. Deletion of the H2A tail sensitizes cells to DNA-damaging agents and PARP inhibitors, implicating a functional role of PARP1-mediated nucleosome disassembly in DNA repair. Together, our findings support a model in which PARP1 directly drives histone eviction, leading to the formation of subnucleosomes that facilitate efficient DNA repair.

Facilitates chromatin transcription (FACT) is a histone chaperone that displaces and re-assembles histones during transcription. Recent studies have reported a minor role for FACT in chromatin architecture. We have recently shown that active gene promoters form nanoscale domains and proposed they are created by the biophysical properties of nucleosome-free regions. Here we use base-pair resolution Micro Capture-C ultra to show that, following FACT degradation, nanoscale domains are lost and subnucleosomal chromatin interactions are rearranged at active promoters. Nucleosome-free regions at these promoters expand and chromatin-binding factors invade the newly accessible chromatin, indicating FACT maintains the integrity of active promoters by opposing DNA-binding factor spreading into gene bodies. Finally, we show increased interactions between promoters across topologically associating domains, suggesting large-scale structural changes upon FACT loss. Thus, we demonstrate FACT plays a major role in chromatin organisation and provide in vivo evidence that nucleosomes drive both local and long-range chromatin architecture.

Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses 1-5. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes 6-10. Whether chromatin marks around individual genes change on these rapid timescales remains unclear. Here we show that local chromatin modification signals around endogenous genes in mouse embryonic stem cells fluctuate reversibly with transcriptional state, using live imaging of individual genes together with fluorescent probes that report histone modifications11-16. Activation-associated acetylation and methylation marks increased in association with transcriptional activation and decreased with inactivation, whereas a Polycomb-associated repressive mark behaved oppositely. Transcriptional coactivators and both histone acetyltransferase and deacetylase complexes were enriched during transcriptionally active state, consistent with opposing enzymatic activities shaping local acetylation levels 17,18. Inhibiting histone deacetylases altered the durations of active and inactive events, supporting a role for deacetylation in regulating transcriptional state transitions. Thus, histone modifications undergo reversible, minute-scale changes coupled to transcriptional activity. This framework helps explain how stochastic transcriptional bursts can occur with stable gene regulation over longer timescales.

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.

Spirochaetota is a deeply rooted bacterial phylum of evolutionary and medical importance. We show that the spirochetal RNA polymerase (RNAP) has a diminished ability to melt promoters compared to its Escherichia coli counterpart. This deficiency is compensated by CarD and is not linked to the phylums natural resistance to the antibiotic rifampicin. CryoEM analysis revealed that the spirochetal RNAP disengages the -35 element during promoter unwinding, which contrasts with other bacterial RNAPs that release this element later during the initial transcription. Our research also details the unusually tight, non-sequence-specific binding of the spirochetal RNAP holoenzyme to DNA, which may be linked to the highly extended structure of the spirochetal nucleoid. We support these observations with a structural analysis of the dimeric holoenzyme non-specifically bound to DNA. Our results highlight the functional diversity of bacterial transcription systems and lay the foundation for understanding transcription regulation in pathogenic spirochetes.

Repressive histone methyltransferases carry a catalytic ("write") domain and a separate domain specialized for recognizing ("reading") the reaction product. This read-write configuration acts as a positive feedback mechanism for epigenetic maintenance and the growth of repressive chromatin domains. Feedback exhibits as catalytic stimulation and is understood to act towards a proximal (trans) nucleosome. Whether this stimulation affects a specific methylation transition and whether it is restricted to trans-stimulation remains opaque. Here, we dissect the positive feedback in the heterodimeric histone 3 lysine 9 (H3K9) mono- and dimethlyase G9a-GLP, which carries two catalytic SET and two product-reading Ankyrin repeat (ANK) domains. We find that reading by both ANK domains is required for H3K9 di-, but not monomethylation on nucleosomes and for tight binding to them. As this read-writing occurs on dilute mononucleosomes, we propose that intranucleosomal feedback occurs for G9a-GLP. Swapping the ANK domains results in loss of dimethylation while maintaining nucleosome binding, indicating catalytic coupling of nucleosome methylation intermediates to reading. Crosslinking mass spectrometry reveals specific G9a surfaces that contact nucleosomal methylation intermediates. Structural approaches reveal how these surfaces position the G9a ANK domain on the methylation-intermediate nucleosome and stabilize G9a-GLP on chromatin during the reaction.

Mono-ubiquitination of histone H2B (H2Bub) is deposited by Bre1-type E3 ubiquitin ligase complexes during transcription elongation and is critical for chromatin organization, DNA repair, and transcription regulation. In S. pombe, this activity is carried out by HULC, a complex of the Bre1 homologs Brl1 and Brl2 with Rhp6 and Shf1. While H2Bub deposition depends on recruitment of HULC to RNA Pol II by the Paf1 complex (PAF1C), the molecular basis for how PAF1C activates HULC has remained unclear. Using AlphaFold modelling, biochemical reconstitution, and functional assays, we define the architecture of HULC as a flexible 1:1:1:1 assembly in which Shf1 stabilizes an elongated coiled-coil hairpin that brings the RING and Rad6-binding domains into proximity. However, for full stimulation of HULC activity, we find that the HMD domain of the PAF1C subunit Prf1 stimulates ubiquitin transfer through a RING-binding region (RBR) that repositions the RING domains adjacent to Rhp6 in a catalytically competent configuration. These findings reveal an activation mechanism in which transcription-associated Prf1 primes HULC for ubiquitin transfer through the Prf1RBR, revealing a critical regulatory interface for H2B ubiquitination.

Ribosome biogenesis and mRNA translation are fundamental cellular processes regulated by a diverse set of protein factors, including GTPases. In bacteria, while several GTPases are known to participate in ribosome assembly, their precise mechanisms in subunit maturation and their potential roles in translation regulation remain largely elusive. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of native pre-50S assembly intermediates and 70S translating ribosomes, isolated via epitope-tagged GTPases from engineered Escherichia coli cells at resolutions of 2.3-4.4 [A]. These structures elucidate how three GTPases, YihA, EngA and ObgE, act as successive placeholders to mediate rRNA folding and to coordinate the correct timing of the maturation of different functional blocks within the large ribosomal subunit. Furthermore, our data identify several previously unrecognized 70S translational complexes bound by the GTPases EngA and BipA--factors traditionally regarded as assembly factors, thereby uncovering their regulatory role in bridging ribosome assembly and translation initiation. Collectively, our findings delineate a GTPase-mediated surveillance system that continuously monitors the assembly of ribosomal subunits and translation adversity, thereby safeguarding protein synthesis and maintaining proteome homeostasis.

Reversible pre-mRNA splicing factor phosphorylation is a well-documented feature of spliceosome assembly, activation, and disassembly, yet its functional and mechanistic roles are still emerging. SF3B1, a core spliceosome subunit, is extensively phosphorylated before the first catalytic step of pre-mRNA splicing. Intriguingly, most SF3B1 phosphorylation sites surround its binding sites for U2AF2, an early-stage pre-mRNA splicing factor. Here, we discovered that SF3B1 phosphorylation significantly decreases its association with U2AF2. We determined two crystal structures revealing electrostatic repulsion between an acidic U2AF2 -helix and the negatively-charged phosphoryl group of SF3B1. Variants with amino acid substitutions that prevent or mimic SF3B1 phosphorylation perturbed thousands of splice sites, primarily those marked by uridine-rich splice site signals recognized by U2AF2. Collectively, our findings demonstrate a widespread but previously unrecognized role for SF3B1 phosphorylation as a gateway for U2AF2 dissociation so that pre-mRNA splicing can proceed.

MacroH2A (mH2A) is a histone variant primarily implicated in heterochromatin maintenance and transcriptional repression, yet how its conserved histone-fold and its variant-specific domains reshape chromatin to enforce gene silencing remains poorly understood. Here, we dissect the domain-specific contributions of mH2A to nucleosome dynamics and chromatin remodeling. We show that, in addition to its linker region, the C-terminal tail of mH2A histone-fold also stabilizes nucleosome entry/exit DNA. Cryo-EM analysis reveals that this C-terminus tracks along nucleosomal DNA toward the dyad, adopting an on-dyad binding mode reminiscent of linker histone H1. Additionally, the mH2A linker potently inhibits DNA translocation by both the INO80 and Chd1 chromatin remodelers, while the histone fold selectively suppresses INO80 activity. In contrast, the macro domain has no detectable impact on terminal DNA accessibility or remodeling. Together, our results uncover a previously unappreciated architectural role for the mH2A histone fold and establish the linker domain as a dominant regulator of nucleosome dynamics and chromatin remodeling, providing a mechanistic framework for how mH2A enforces transcriptional repression.

We report the discovery and structure of a previously unknown ~1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.