Santos J, Günnigmann M, Gora RJ
… +13 more, Iljina M, Predin M, Kotan IE, De P, Choudhary D, Jang J, Tippmann F, Hins C, Ban N, Tans SJ, Shan SO, Kramer G, Bukau B
The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While the role of NAC as a dynamic hub for other factors is well establish...The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While the role of NAC as a dynamic hub for other factors is well established, its direct role in co-translational folding is unclear. By proteome-wide profiling of co-translational NAC interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and α-helical propensity within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its β-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and in mitochondrial membrane protein biogenesis. Together, these findings show that human NAC acts as a bona fide co-translational chaperone that directly promotes early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.
Replicative single-stranded DNA gaps are emerging as central intermediates in the cellular response to replication stress. Replication frequently continues past lesions or difficult-to-replicate regions through leading-s...Replicative single-stranded DNA gaps are emerging as central intermediates in the cellular response to replication stress. Replication frequently continues past lesions or difficult-to-replicate regions through leading-strand repriming or delayed Okazaki fragment (OKF) maturation, generating structured gaps requiring stabilization and repair. Here, we describe the major routes of gap formation, including polymerase-helicase uncoupling, impaired OKF processing, PrimPol-mediated lesion bypass, and endogenous abasic site accumulation from base excision repair and DNA methylation turnover. We examine the mechanisms that suppress, protect, and resolve these gaps, highlighting RAD51/BRCA2-mediated stabilization, PCNA modifications, PARP1- and CTC1-STN1-TEN1 (CST)-dependent fill-in pathways, and the balance between translesion synthesis and template switching. Finally, we discuss how persistent gaps drive fork degradation, genome instability, and innate immune activation, contributing to explaining the therapeutic vulnerabilities and resistance of cancer cells to PARP, polymerase Q (Pol θ), and ATR inhibitors. This perspective presents a unified model in which timely replicative gap recognition and resolution ensure genome stability.
In eukaryotes, incompletely and aberrantly processed mRNAs as well as numerous noncoding RNAs are retained in the nucleus and often degraded, but the underlying quality-control mechanisms remain poorly defined. Here, we...In eukaryotes, incompletely and aberrantly processed mRNAs as well as numerous noncoding RNAs are retained in the nucleus and often degraded, but the underlying quality-control mechanisms remain poorly defined. Here, we identify LENG8 as a conserved RNA nuclear retention and degradation factor. LENG8 is recruited to pre-mRNAs by splicing factors, including the U1 small nuclear ribonucleoprotein particle (snRNP). It associates with PCID2 and SEM1 to form the REX (repressor of export) complex, which is conserved from yeast to humans, and causes RNA nuclear retention by acting as a dominant-negative factor for the essential mRNA export factor TREX (transcription-export)-2. Loss of LENG8 results in cytoplasmic leakage of misprocessed mRNAs, including intronically polyadenylated and intron-retained mRNAs, as well as noncoding RNAs. Moreover, LENG8 promotes nuclear RNA degradation through interactions with the RNA exosome adaptor PAXT. Together, these findings uncover a conserved RNA quality-control mechanism that ensures only correctly processed RNAs are exported.
Yuan C, Jiang J, Cheng Y
… +14 more, Lian JP, Liao YH, Yu Y, Zhao WL, Mo WT, Yang JB, Qin YC, Chen ZT, Yang L, Lei MQ, Zhang YC, He RR, Zhou YF, Chen YQ
Lnc-eRNAs are long non-coding RNAs (lncRNAs) whose transcription initiation sites overlap with enhancers, but whether lnc-eRNAs exist in plants and whether they affect disease resistance remain unknown. Here, we identifi...Lnc-eRNAs are long non-coding RNAs (lncRNAs) whose transcription initiation sites overlap with enhancers, but whether lnc-eRNAs exist in plants and whether they affect disease resistance remain unknown. Here, we identified lnc-eRNAs in rice (Oryza sativa L.) using transcriptome and chromatin accessibility data and characterized one lnc-eRNA, Xanthomonas Susceptible Enhancer RNA1 (XSER1), which was quickly activated under pathogen infection. XSER1 knockdown conferred resistance to bacterial and fungal diseases caused by Xanthomonas and Magnaporthe oryzae, respectively, showing broad-spectrum disease resistance. We found that XSER1 binds to the transcription factor basic helix-loop-helix 94 (OsbHLH94) and promotes chromatin looping to shape chromatin accessibility and rewire XSER1 activation, as well as affecting the expression of the adjacent histone H3 lysine 4 (H3K4) demethylase gene JUMONJI 707 (OsJMJ707), thus conferring resistance to pathogens. XSER1 abundance is very low under normal conditions but increases substantially within hours of pathogen infection. Knocking out XSER1 did not cause developmental defects but enhanced plant defenses, revealing its potential for breeding pathogen-resistant plants.
In a recent issue of Science, Chao et al. describe TimeVault, an approach that allows researchers to reconstruct past transcriptional states even as cells continue to divide and differentiate. It provides an alternative...In a recent issue of Science, Chao et al. describe TimeVault, an approach that allows researchers to reconstruct past transcriptional states even as cells continue to divide and differentiate. It provides an alternative to conventional single-cell RNA sequencing (scRNA-seq), which captures only a terminal "snapshot," by preserving molecular history within intact cells.
In this issue of Molecular Cell, Song et al. focus on the regulation of hypoxia-induced alternative splicing (AS) in cancer cells and provide evidence for key regulatory roles for MALAT1-SRSF1 condensates in preferential...In this issue of Molecular Cell, Song et al. focus on the regulation of hypoxia-induced alternative splicing (AS) in cancer cells and provide evidence for key regulatory roles for MALAT1-SRSF1 condensates in preferential pre-mRNA binding and RNAPII recruitment.
Extensive transcription of genetic information beyond annotated genes is an asset for cellular physiology and evolution, but it can blur functional messages and threaten genomic stability. In this issue of Molecular Cell...Extensive transcription of genetic information beyond annotated genes is an asset for cellular physiology and evolution, but it can blur functional messages and threaten genomic stability. In this issue of Molecular Cell, Polizzese et al. reveal how transcriptional brakes act specifically on non-coding sequences through nascent RNA recognition by the Restrictor.
In this issue, Yuan et al. identify a pathogen-inducible long noncoding enhancer RNA (lnc-eRNA), XSER1, that regulates rice resistance by promoting chromatin looping to regulate transcription of a nearby gene. This study...In this issue, Yuan et al. identify a pathogen-inducible long noncoding enhancer RNA (lnc-eRNA), XSER1, that regulates rice resistance by promoting chromatin looping to regulate transcription of a nearby gene. This study establishes lnc-eRNA as a functional regulator in plant immunity, linking enhancer RNA, transcriptional regulation, chromatin looping, and accessibility.
Squair DR, Rivers E, Sowar H
… +12 more, Balci A, Harmo R, Wright DJ, Beniwal G, Soetens M, Mathur S, Tollervey A, Wood NT, Pao KC, Stanton C, Fletcher AJ, Virdee S
The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion, and RNA sequestration. Mutations in the gene zinc-finger NFX1-type c...The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion, and RNA sequestration. Mutations in the gene zinc-finger NFX1-type containing 1 (ZNFX1) cause severe pediatric immunodeficiencies, including chronic viral infection and autoinflammation. Here, we show that ZNFX1 is an RNA helicase with cryptic and unusual bifurcating E3 ubiquitin ligase activity. Nucleotide-dependent RNA binding stimulates ZNFX1 to generate complex ubiquitin chains via a two-component ubiquitin circuit wired in parallel, with ubiquitin flux occurring via two competing paths. One route produces K63-linked polyubiquitin that drives RNA entrapment within self-propagating ZNFX1 aggregates, and the other route produces K48-linked polyubiquitin that drives ZNFX1 turnover. RNA entrapment restricts RNA virus replication and is reversible by deubiquitination. Pathogenic ZNFX1 variants are defective for viral restriction, linking RNA entrapment to antiviral immunity in vivo.
Micronuclei are membrane-encapsulated nuclear aberrations that form following chromosome segregation errors. Micronuclear membrane collapse permits access of the pattern recognition receptor cGAS and its antagonist, the...Micronuclei are membrane-encapsulated nuclear aberrations that form following chromosome segregation errors. Micronuclear membrane collapse permits access of the pattern recognition receptor cGAS and its antagonist, the TREX1 exonuclease. TREX1 endoplasmic reticulum tethering is essential for invasion into ruptured micronuclei, but the mechanisms underlying this dependency are unknown. Here, we identify BAF as a key regulator of TREX1 activity at micronuclei. BAF accumulates at micronuclei following membrane collapse and augments TREX1 recruitment via interactions with LEM-domain proteins. Despite delayed entry, TREX1 exhibits enhanced micronuclear DNA degradation and independence from ER-tethering in BAF-deficient cells. Recombinant BAF inhibits TREX1-mediated DNA degradation in vitro via DNA-binding. BAF similarly outcompetes cGAS for micronuclear DNA and limits cGAS-dependent immune signaling. These findings reveal a BAF-dependent protective barrier to the diffusive entry of DNA-binding proteins into ruptured micronuclei that explains TREX1 ER-tethering requirements for suppressing innate immune responses in chromosomally unstable cells.
Song YJ, Shinn MK, Bangru S
… +13 more, Wang Y, Sun Q, Hao Q, Chaturvedi P, Freier SM, Perez-Pinera P, Nelson ER, Belmont AS, Guttman M, Prasanth SG, Kalsotra A, Pappu RV, Prasanth KV
The alternative splicing (AS) of pre-mRNA regulates key cellular processes, and its dysregulation is linked to tumorigenesis. Hypoxia, a common feature of malignant tumors, triggers AS in thousands of genes. The mechanis...The alternative splicing (AS) of pre-mRNA regulates key cellular processes, and its dysregulation is linked to tumorigenesis. Hypoxia, a common feature of malignant tumors, triggers AS in thousands of genes. The mechanisms controlling hypoxia-responsive AS remain unclear. We observe that hypoxia-responsive spliced exons exhibit characteristics of inefficient splicing, and the genes encoding these transcripts are pre-positioned near nuclear speckles-the membraneless nuclear bodies that boost splicing. The speckle-enriched long noncoding RNA (lncRNA) MALAT1 (Metastasis-associated lung adenocarcinoma transcript 1), induced during hypoxia, associates with the hypoxia-responsive genes. Furthermore, MALAT1 promotes AS by modulating the interaction between the SR family of splicing factor 1 (SRSF1) and pre-mRNAs. Mechanistically, MALAT1 promotes the condensation of SRSF1, and the condensates are preferentially recognized and recruited by RNA polymerase II (RNAPII). Overall, our results demonstrate that MALAT1 dictates hypoxia-induced AS by organizing splicing factor condensates near speckles to enable the efficient RNAPII-mediated recruitment of splicing factors to pre-mRNAs.
Li S, Zhao Y, Li Y
… +14 more, Shah SB, Shi Y, Nguyen T, Wang Z, Chang CY, Ray A, Bu TH, Loguercio S, Sasaki T, Sussman JH, Wang H, Gilbert DM, Aladjem MI, Wu X
Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replic...Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. While promoted by DNA polymerase theta (Polθ), fork-MMEJ operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, distinct from canonical MMEJ (cMMEJ), which is defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. The combined inactivation of ATR and Polθ synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.
The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), the guide RNA (tigRNA)...The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), the guide RNA (tigRNA) of the tandem interspaced guide RNA (TIGR) system features a dual-spacer arrangement, thereby directing the TIGR-associated (Tas) protein to engage both strands of the target double-stranded DNA (dsDNA). Here, we determine six cryo-electron microscopy structures of the Salicola phage TIGR-TasH complex. The central coiled-coil region of TasH mediates dimerization, while the C-terminal nucleolar protein (Nop) domain is able to autonomously process precursor tigRNA. Upon target binding, the dynamic N-terminal HNH nuclease domain is recruited for cleavage through a β-hairpin, which also determines the target preference. More interestingly, the conserved box C motif of tigRNA stabilizes this β-hairpin in an adenine-specific manner, enabling us to rationally design a guide RNA-defined nickase, distinct from conventional protein-based nickase strategies used in genome editing.
Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties and the processes they impact remain poorly understood, esp...Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties and the processes they impact remain poorly understood, especially in the nucleus. We hypothesized that some viruses might change the biophysical properties of the nucleus to favor virus survival and replication and found that herpes simplex virus 1 (HSV-1) increases the mesoscale fluidity of the nucleus. The HSV-1 protein ICP4 (infected cell protein 4) caused fluidization and enabled the growth of synthetic nuclear condensates. Conversely, conditions that decreased nuclear fluidity inhibited the growth of viral replication compartment condensates and reduced infectious virus production. Together, our data suggest that ICP4 increases nuclear fluidity to promote the formation of condensates that drive the progression of the HSV-1 life cycle. We speculate that a key function of ICP4 is to overcome the crowding and elastic confinement within cell nuclei that are a fundamental barrier to virus replication.
Phase separation is increasingly recognized in facultative heterochromatinization of Polycomb target genes; however, the mechanisms underlying this process remain obscure. Using single-molecule imaging and tracking, we s...Phase separation is increasingly recognized in facultative heterochromatinization of Polycomb target genes; however, the mechanisms underlying this process remain obscure. Using single-molecule imaging and tracking, we show that individual condensates in mouse embryonic stem cells (mESCs) contain approximately 3 CBX2 molecules and numerous Polycomb repressive complex (PRC)1 and PRC2 subunits and indicate that the composition and dynamics of condensates are developmentally regulated. We reveal that CBX2 clusters PRC2 and controls the spatial distribution of both PRC2 and H3K27me3. Using genomic approaches, we demonstrate that CBX2 binds to condensate initiation sites, which are enriched for PRC2 nucleation sites. CBX2 deletion causes PRC2 and H3K27me3 to redistribute from their regular targets. By developing a separation-of-function variant, we determine that CBX2 relies on its self-clustering ability to function. These findings collectively support a phase-separation model driven by nucleation and bridging, in which low-abundance proteins self-cluster to initiate condensate assembly, a process tightly coupled to function.
Zhu K, Li WC, Liu J
… +19 more, Yang BA, Guzman E, Guzman M, Sit R, Yan J, Granat A, Chen SA, Parekh U, Pulos-Holmes M, Ouyang Y, Dietzel C, Nielsen SCA, Yamazaki M, Mulas C, Minopoli R, Tyanova S, Itzhak DN, Acosta-Alvear D, Chen J
The human genome harbors thousands of unannotated short open reading frames (sORFs) with the potential to encode microproteins, yet their physiological roles remain largely unexplored. Here, we developed sORF-seq, a func...The human genome harbors thousands of unannotated short open reading frames (sORFs) with the potential to encode microproteins, yet their physiological roles remain largely unexplored. Here, we developed sORF-seq, a functional screen that identified hundreds of sORF-encoded microproteins regulating cellular differentiation. Among these, we discovered lncPRESS1 as a critical regulator of cell fate, and remarkably, it acts as a bifunctional RNA. In the nucleus, lncPRESS1 functions as a long non-coding RNA (lncRNA), guiding the genomic distribution of SWI/SNF and orchestrating developmental gene expression programs. In the cytoplasm, lncPRESS1 acts as an mRNA, translated into a microprotein that directs lineage commitment through Sonic hedgehog (SHH) signaling pathways and interactions with the primary cilium. This dual functionality allows lncPRESS1 to coordinate nuclear and cytoplasmic regulatory networks, shaping early embryogenesis and human brain development. Our findings unveil an unexpected paradigm of non-canonical ORFs in choreographing complex pathways, expanding our understanding of the functional genome beyond traditional coding genes.
Agius SC, Boudes M, Healy E
… +11 more, Kim Y, Chang YC, Murray M, Manent J, Rientjes J, Lai Z, Nixon S, Homman-Ludiye J, Levina V, McGlinn E, Davidovich C
The Polycomb repressive complex 2 (PRC2) is essential for normal development by maintaining developmental gene repression. PRC2 deposits the repressive chromatin mark H3 lysine 27 tri-methyl (H3K27me3) through a read-wri...The Polycomb repressive complex 2 (PRC2) is essential for normal development by maintaining developmental gene repression. PRC2 deposits the repressive chromatin mark H3 lysine 27 tri-methyl (H3K27me3) through a read-write loop that involves direct interactions between PRC2 and H3K27me3. According to current models, the PRC2-H3K27me3 read-write loop is initiated by the PRC2 subunits JARID2 and PALI1 that mimic H3K27me3. However, it is unknown what restricts the PRC2-H3K27me3 read-write loop from spreading H3K27me3 indefinitely. To answer this question, we generated mutant mice where PRC2 subunits cannot mimic H3K27me3. Unexpectedly, the mutations led to delayed Hox gene activation and a homeotic transformation characteristic of a Polycomb gain of function in vivo and the spread of H3K27me3 beyond Polycomb domains in stem cells. Collectively, we show that H3K27me3 mimicry evolved to compete against the PRC2-H3K27me3 read-write loop in a process that restrains PRC2 and restricts the spread of Polycomb domains.
Mammalian genomes harbor hundreds of thousands of RNA polymerase II (RNA Pol II) landing pads, enhancers, and promoters from which transcription initiates bidirectionally. Nevertheless, processive transcription is largel...Mammalian genomes harbor hundreds of thousands of RNA polymerase II (RNA Pol II) landing pads, enhancers, and promoters from which transcription initiates bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. An essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive RNA Pol II activity at extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the mechanistic basis for its selectivity for extragenic transcription is unclear. Here, we show that while WDR82 tethers Restrictor to transcription initiation sites, the C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with motifs enriched at the 5' end of extragenic transcripts, with such interactions being required for transcription termination. Hence, although Restrictor recruitment requires WDR82-dependent tethering to the initiating RNA Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Nascent polypeptide chains emerging from the ribosome engage a range of co-translational factors at distinct phases of translation. These co-translational interactions are crucial for proper protein biogenesis and qualit...Nascent polypeptide chains emerging from the ribosome engage a range of co-translational factors at distinct phases of translation. These co-translational interactions are crucial for proper protein biogenesis and quality control pathways to maintain protein homeostasis. Hence, the systematic identification of these co-translational interactors provides insights into how distinct polypeptide fates are determined. Here, we developed nascent-chain interactor profiling (NCIP), a metabolic-labeling- and chemical-crosslinking-enabled proteomics method to identify proteins interacting with nascent polypeptide chains at a proteome-wide scale. Results from NCIP support the co-translational assembly model of multiple protein complexes and reveal TRIM25 as a co-translational E3 ubiquitin ligase. TRIM25 ubiquitinates misfolded nascent chains for quality control at the ribosome. Our results provide a generalizable framework to systematically profile co-translational interactors.
Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functio...Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.