Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

A small nucleosome from a weird virus with a fat genome.

Valencia-Sánchez et al. (2021) and Liu et al. (2021) provide structural and biological insights about the existence and importance of a nucleosome-like particle in a family of giant viruses.

Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA.

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.

Molecular basis of RNA polymerase III transcription repression by Maf1.

RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.

Structural basis of RNA polymerase III transcription initiation.

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4-4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.

Condensin complexes: understanding loop extrusion one conformational change at a time.

Condensin and cohesin, both members of the structural maintenance of chromosome (SMC) family, contribute to the regulation and structure of chromatin. Recent work has shown both condensin and cohesin extrude DNA loops and most likely work via a conserved mechanism. This review focuses on condensin complexes, highlighting recent in vitro work characterising DNA loop formation and protein structure. We discuss similarities between condensin and cohesin complexes to derive a possible mechanistic model, as well as discuss differences that exist between the different condensin isoforms found in higher eukaryotes.

Conservation between the RNA polymerase I, II, and III transcription initiation machineries.

Recent studies of the three eukaryotic transcription machineries revealed that all initiation complexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved core initiation complex stabilizes the open DNA promoter complex and directs initial RNA synthesis. The periphery of the core initiation complex is decorated by additional polymerase-specific factors that account for functional differences in promoter recognition and opening, and gene class-specific regulation. This review outlines the similarities and differences between these important molecular machines.

A structural perspective on RNA polymerase I and RNA polymerase III transcription machineries.

RNA polymerase I and III are responsible for the bulk of nuclear transcription in actively growing cells and their activity impacts the cellular biosynthetic capacity. As a consequence, RNA polymerase I and III deregulation has been directly linked to cancer development. The complexity of RNA polymerase I and III transcription apparatuses has hampered their structural characterization. However, in the last decade tremendous progresses have been made, providing insights into the molecular and functional architecture of these multi-subunit transcriptional machineries. Here we summarize the available structural data on RNA polymerase I and III, including specific transcription factors and global regulators. Despite the overall scarcity of detailed structural data, the recent advances in the structural biology of RNA polymerase I and III represent the first step towards a comprehensive understanding of the molecular mechanism underlying RNA polymerase I and III transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols.

RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges because of subsequent waves and long-term consequences of great concern. Here, we chart the molecular basis of COVID-19 pathogenesis by analyzing patients' immune responses at single-cell resolution across disease course and severity. This approach confirms cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identifies a severity-associated activation of the receptor for advanced glycation endproducts (RAGE) pathway in monocytes. In vitro THP1-based experiments indicate that monocytes bind the SARS-CoV-2 S1-receptor binding domain (RBD) via RAGE, pointing to RAGE-Spike interaction enabling monocyte infection. Thus, our results demonstrate that RAGE is a functional receptor of SARS-CoV-2 contributing to COVID-19 severity.

The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis.

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40-370. While the N-terminal part of that minimal region (residues 47-247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248-370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.

Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II.

RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization.

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes.

Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.

A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component.

Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity, with an abundant chromatin-associated factor, is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe.

MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface.

Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin's loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis.