Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

Structural insights into nuclear transcription by eukaryotic DNA-dependent RNA polymerases.

The eukaryotic transcription apparatus synthesizes a staggering diversity of RNA molecules. The labour of nuclear gene transcription is, therefore, divided among multiple DNA-dependent RNA polymerases. RNA polymerase I (Pol I) transcribes ribosomal RNA, Pol II synthesizes messenger RNAs and various non-coding RNAs (including long non-coding RNAs, microRNAs and small nuclear RNAs) and Pol III produces transfer RNAs and other short RNA molecules. Pol I, Pol II and Pol III are large, multisubunit protein complexes that associate with a multitude of additional factors to synthesize transcripts that largely differ in size, structure and abundance. The three transcription machineries share common characteristics, but differ widely in various aspects, such as numbers of RNA polymerase subunits, regulatory elements and accessory factors, which allows them to specialize in transcribing their specific RNAs. Common to the three RNA polymerases is that the transcription process consists of three major steps: transcription initiation, transcript elongation and transcription termination. In this Review, we outline the common principles and differences between the Pol I, Pol II and Pol III transcription machineries and discuss key structural and functional insights obtained into the three stages of their transcription processes.

Pervasive Protein Thermal Stability Variation during the Cell Cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

The mission to ensure continued funding for excellent basic research.

The surprising decision by Novo Nordisk Foundation (NNF) to discontinue funding for the Center for Protein Research in Copenhagen should prompt discussions about public and private commitment to support basic research.

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage.

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

Molecular mechanism of promoter opening by RNA polymerase III.

RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures of Pol III preinitiation complexes, comprising the 17-subunit Pol III and the heterotrimeric transcription factor TFIIIB, bound to a natural promoter in different functional states. Electron cryo-microscopy reconstructions, varying from 3.7 Å to 5.5 Å resolution, include two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system.

Molecular Structures of Transcribing RNA Polymerase I.

RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.

Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1.

In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

Structural insights into transcription initiation by yeast RNA polymerase I.

In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

Molecular structures of unbound and transcribing RNA polymerase III.

Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements.

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.

Automated structure modeling of large protein assemblies using crosslinks as distance restraints.

Crosslinking mass spectrometry is increasingly used for structural characterization of multisubunit protein complexes. Chemical crosslinking captures conformational heterogeneity, which typically results in conflicting crosslinks that cannot be satisfied in a single model, making detailed modeling a challenging task. Here we introduce an automated modeling method dedicated to large protein assemblies ('XL-MOD' software is available at http://aria.pasteur.fr/supplementary-data/x-links) that (i) uses a form of spatial restraints that realistically reflects the distribution of experimentally observed crosslinked distances; (ii) automatically deals with ambiguous and/or conflicting crosslinks and identifies alternative conformations within a Bayesian framework; and (iii) allows subunit structures to be flexible during conformational sampling. We demonstrate our method by testing it on known structures and available crosslinking data. We also crosslinked and modeled the 17-subunit yeast RNA polymerase III at atomic resolution; the resulting model agrees remarkably well with recently published cryoelectron microscopy structures and provides additional insights into the polymerase structure.

Architecture of the yeast Elongator complex.

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.

Crystal structure of the 14-subunit RNA polymerase I.

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49-A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

Molecular architecture of polycomb repressive complexes.

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin.

An integrated approach for genome annotation of the eukaryotic thermophile Chaetomium thermophilum.

The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics data, and integrated them with computational annotation methods and a multitude of biochemical experiments conducted by the structural biology community. We considerably improved the genome annotation of Chaetomium thermophilum and characterized the transcripts and expression of thousands of genes. We furthermore show that the composition and structure of the expressed proteome of Chaetomium thermophilum is similar to its mesophilic relatives. Data were deposited in a publicly available repository and provide a rich source to the structural biology community.