Molecular Cell最新文献

RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.

Replication stress is a key driver of DNA damage and genome instability. Here, we report that replication stress induces an inflammatory response in the absence of DNA damage. The DNA-sensing factor interferon-γ-inducible factor 16 (IFI16) binds nascent DNA at stalled replication forks and signals via the adaptor stimulator of interferon genes (STING) to induce activation of nuclear factor κB (NF-κB) and the production of pro-inflammatory cytokines, independently of the cytosolic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). Replication stress-induced fork remodeling generates a new DNA end that is vulnerable to degradation by nucleases and is protected by a range of factors, including the tumor suppressors BRCA1 and BRCA2. IFI16 acts directly at stalled replication forks to protect nascent DNA from degradation by the nucleases MRE11, EXO1, and DNA2. Furthermore, IFI16 is required for the interferon-mediated rescue of fork protection in BRCA-deficient cells, highlighting the critical role of IFI16 in the crosstalk between innate immunity and fork protection during replication stress.

Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA polymerase II (RNA Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed for disease therapeutics. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation control, and mechanisms by which other BET proteins regulate RNA Pol II remain insufficiently understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cell lines. We demonstrate that BRD2 bromodomains preferentially bind to histone H4 harboring MOF-mediated H4K16ac, while the BRD2 C-terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into distinct roles for BRD2 and BRD4 in transcriptional initiation and elongation control for proper regulation of gene expression.

Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, in which a newly synthesized positive strand replaces the parental positive strand, which is released as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus ɸ6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.