Kim H, Kim DG, Ha S
… +17 more, Jang JY, Vitorino FNL, Gongora JM, Kim W, Oh Y, Park G, Kang H, Im SH, Jung CK, Kim SY, Jung I, Won JK, Garcia BA, Kim SJ, Lee KE, Ryu JK, Lee CH
Dysregulation of Polycomb Repressive Complex 2 (PRC2) contributes to cancer. Of its catalytic subunits, Enhancer of Zeste (EZH) 1 and EZH2, EZH2 mutations are extensively studied, but the role of EZH1 in cancer remains l...Dysregulation of Polycomb Repressive Complex 2 (PRC2) contributes to cancer. Of its catalytic subunits, Enhancer of Zeste (EZH) 1 and EZH2, EZH2 mutations are extensively studied, but the role of EZH1 in cancer remains largely unexplored. Here, we investigate the thyroid cancer-associated EZH1 mutation and uncover a mechanism that extends beyond catalytic gain of function. Using biochemical, single-molecule, epigenomic, and transcriptomic analyses, we demonstrated that EZH1 significantly enhances chromatin compaction and stimulates PRC2-EZH1 catalytic activity, thereby rewiring PRC2-chromatin interactions. This altered engagement enables PRC2 activity within H3K36me2-marked chromatin typically refractory to H3K27 methylation, leading to widespread epigenetic and transcriptional reprogramming. Notably, enhanced chromatin compaction is observed with EZH1 but not with the corresponding EZH2 mutation, indicating an EZH1-specific mechanism. Functionally, EZH1 accelerated tumor growth in vivo and induced pronounced micronuclei formation ex vivo, reflecting aberrant chromatin compaction and genome instability. Together, our study demonstrates that EZH1 promotes a permissive epigenomic landscape for the progression of follicular thyroid cancer.
How transcription factors (TFs) and their binding sites organize and engage nucleosomes at natural genomic locations remains poorly understood. Here, we develop Benzonase-seq to measure the rotational phasing of nucleoso...How transcription factors (TFs) and their binding sites organize and engage nucleosomes at natural genomic locations remains poorly understood. Here, we develop Benzonase-seq to measure the rotational phasing of nucleosomes in human cells and enhance chromatin immunoprecipitation (ChIP)-exo (v6) to measure rotational phasing on the same DNA molecule bound by a TF. Unbound CTCF sites were found to be rotationally accessible on nucleosomes, and this rotational accessibility is encoded by classical dinucleotide periodicities. CTCF binding results in nucleosome displacement to adjacent DNA phasing sequences. Upon examining 40 TF classes, unbound sites were found to be phased either inward or outward or to lack phasing. In all examined cases, TF binding (e.g., NFIA and FoxA) results in adjacent rotational and translational phasing, which is not dinucleotide encoded. Benzonase-seq also more robustly maps nucleosome and subnucleosome positions in hard-to-map CpG islands. These findings provide a clearer view of how TFs engage and position nucleosomes to shape the natural chromatin landscape.
Precise intron removal by RNA splicing is essential for faithful gene expression, yet the mechanisms ensuring splicing fidelity in mammals remain unclear. Using a systematic knockdown RNA sequencing (RNA-seq) screen, we...Precise intron removal by RNA splicing is essential for faithful gene expression, yet the mechanisms ensuring splicing fidelity in mammals remain unclear. Using a systematic knockdown RNA sequencing (RNA-seq) screen, we uncover widespread splicing errors and identify AQR, SYF1, and SYF3 as cooperative safeguards of 3' splice-site (3'ss) fidelity in human and mouse. These factors act during spliceosome assembly to correct U2AF-mediated misrecognition of non-canonical 3'ss bearing AG dinucleotides embedded within pyrimidine-rich sequences and lacking canonical branch points (BPs), likely through kinetic proofreading. Their loss triggers pervasive 3'ss mis-splicing, resulting in the accumulation of misfolded proteins, proteotoxic stress, unfolded protein response activation, and ultimately cell death and intestinal inflammation. Together, our study reveals a previously unrecognized layer of splicing fidelity control in mammals that links aberrant splice-site selection to proteostasis and inflammation.
Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that tri-methylates histone H3K27 as the catalytic subunit of the polycomb repressive complex 2. However, inhibition of EZH2 methyltransferase showed only variabl...Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that tri-methylates histone H3K27 as the catalytic subunit of the polycomb repressive complex 2. However, inhibition of EZH2 methyltransferase showed only variable anti-cancer efficacy, suggesting that this approach is insufficient. Here, we demonstrate a methyltransferase-independent mechanism of EZH2 wherein EZH2 interacts with inosine monophosphate dehydrogenase 2 (IMPDH2) in the cytoplasm to promote guanosine-5'-triphosphate (GTP) synthesis. Mass spectrometry identified methyltransferase-independent interactions between the EED-binding domain of EZH2 and the CBS domain of IMPDH2. EZH2 knockdown impeded IMPDH2 and reduced GTP levels, ribosome biogenesis, and cancer progression-effects reversed by guanosine. IMPDH2 knockout antagonized EZH2's tumor-promoting effects in vivo, and increased cytosolic EZH2 and IMPDH2 expression was observed in human melanomas and associated with nucleolar enlargement. EZH2-IMPDH2 complexes were also observed across multiple cancers, wherein Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic. These findings reveal a methyltransferase-independent oncogenic mechanism of EZH2.
In a recent issue of Science, Deng et al. report a bacterial defense-associated reverse transcriptase, DRT3, that synthesizes poly(GT/AC) repeat double-stranded DNA by coupling RNA-templated and protein-templated mechani...In a recent issue of Science, Deng et al. report a bacterial defense-associated reverse transcriptase, DRT3, that synthesizes poly(GT/AC) repeat double-stranded DNA by coupling RNA-templated and protein-templated mechanisms, expanding the functional repertoire of reverse transcriptases in antiviral immunity.
In this issue of Molecular Cell, Chen et al. introduce Ribo-Tweezer: a modular platform for conditionally and selectively depleting proteins from mature ribosomes, enabling direct tests of their ribosome-bound roles in t...In this issue of Molecular Cell, Chen et al. introduce Ribo-Tweezer: a modular platform for conditionally and selectively depleting proteins from mature ribosomes, enabling direct tests of their ribosome-bound roles in translation regulation.
In this issue, Ahsan et al. reveal that de novo lipogenesis averts biguanide-induced reductive stress by consuming reduced nicotinamide adenine dinucleotide phosphate, reframing fatty acid synthesis as a conserved redox...In this issue, Ahsan et al. reveal that de novo lipogenesis averts biguanide-induced reductive stress by consuming reduced nicotinamide adenine dinucleotide phosphate, reframing fatty acid synthesis as a conserved redox safety valve that determines whether biguanide-driven metabolic stress remains adaptive or turns lethal.
In this issue of Molecular Cell, Zhou et al. report that pioneer transcription factor Ascl1-E12a exploits nucleosomes as active association platforms, using separable histone-contacting modes to drive stepwise DNA unwrap...In this issue of Molecular Cell, Zhou et al. report that pioneer transcription factor Ascl1-E12a exploits nucleosomes as active association platforms, using separable histone-contacting modes to drive stepwise DNA unwrapping and repositioning for chromatin opening and neural reprogramming.
Proteostasis is essential for cellular function, and its dysregulation underlies a wide spectrum of diseases. Growing evidence underscores liquid-liquid phase separation (LLPS) as a central mechanism governing protein de...Proteostasis is essential for cellular function, and its dysregulation underlies a wide spectrum of diseases. Growing evidence underscores liquid-liquid phase separation (LLPS) as a central mechanism governing protein degradation through the formation of condensates for proteostasis. These membraneless biomolecular condensates concentrate or sequester key degradation factors, substrates, and enzymes, enabling spatiotemporally regulated protein clearance. Condensates for proteostasis represent a mechanism for degrading pathogenic proteins that remain refractory to conventional therapeutics. In this perspective, we explore how LLPS drives the assembly of condensates for proteostasis, outline their physiological functions, and highlight their emerging utility as a versatile platform for targeted protein degradation. Harnessing these condensates offers a promising route to eliminate "undruggable" targets and reestablish proteostasis, opening new avenues for precision medicine across a range of major diseases.
How mitochondrial respiration is tightly regulated by energy demand remains incompletely defined. When mammalian cells switch from glucose to galactose as a carbon source, we observed the enhanced assembly of respiratory...How mitochondrial respiration is tightly regulated by energy demand remains incompletely defined. When mammalian cells switch from glucose to galactose as a carbon source, we observed the enhanced assembly of respiratory chain complexes accompanied by a marked reduction in TMEM141, a mitochondrial inner membrane protein. Loss of TMEM141 increased mitochondrial respiration and promoted complex I assembly, whereas galactose-induced complex I assembly was markedly blunted in TMEM141-deficient cells. TMEM141 interacts with the complex I assembly factor TIMMDC1, limiting its association with complex I subunits. TMEM141 is degraded by the mitochondrial proteases AFG3L2 and YME1L1, and galactose treatment strengthens their interactions. TMEM141 deficiency increases oxidative damage and mtDNA release, leading to activation of the cGAS-STING pathway. In Drosophila, dTMEM141 localizes to mitochondria, modulates mitochondrial activity, and is required for glial cell integrity in the eye. Together, our findings reveal TMEM141 as a negative regulator of complex I assembly that adapts to oxidative phosphorylation (OXPHOS) demands.
Jagannatha P, Yoon Y, Landry SB
… +22 more, Naritomi JT, Zhan L, Olson S, Wei X, Street LA, Liu L, Jeong J, Quan C, Reid J, Soles LV, Segeberg M, Schmok JC, Jain M, Rosales EKF, Chen VW, Gosztyla ML, Xu S, Pong A, Jovanovic M, Graveley BR, Shi Y, Yeo GW
Alternative polyadenylation (APA) generates transcript isoforms with distinct 3' ends, yet the repertoire of its protein regulators remains poorly defined. Using a large-scale tethered function screen, we profiled 879 hu...Alternative polyadenylation (APA) generates transcript isoforms with distinct 3' ends, yet the repertoire of its protein regulators remains poorly defined. Using a large-scale tethered function screen, we profiled 879 human RNA-binding proteins (RBPs) and identified 63 high-confidence activators of poly(A) site (PAS) selection, most of which were not previously linked to APA. We validated these factors by knockdown PAS-seq, RNA sequencing (RNA-seq), and enhanced cross-linking and immunoprecipitation (eCLIP) analyses and developed a fine-tuned protein language model that predicts PAS selection activators and their key functional domains. We then mechanistically dissected two unexpected hits: GRB2, a signaling adaptor protein, and RNPS1, a peripheral component of the exon junction complex (EJC). Both regulate APA, at least in part, through direct interactions with distinct subunits of the cleavage and polyadenylation (CPA) machinery. Together, our study provides a comprehensive resource of APA-regulating RBPs and uncovers unexpected roles of signaling and EJC factors in APA regulation.
Most existing RNA sequencing methods rely on cell lysis or fixation, limiting their use in longitudinal studies of the same cell population. Here, we introduce POND-seq (protein nanocage-empowered non-destructive sequenc...Most existing RNA sequencing methods rely on cell lysis or fixation, limiting their use in longitudinal studies of the same cell population. Here, we introduce POND-seq (protein nanocage-empowered non-destructive sequencing), a strategy that employs secretory protein nanocages fused to RNA-binding proteins (RBPs) to recover RBP-associated RNAs from living cells. POND-seq robustly identifies RNA targets of cytoplasmic RBPs across multiple cell types and enables longitudinal tracking of dynamic changes in RBP-associated RNA profiles under stress conditions. Fusion to poly(A)-binding protein C (PABPC1) further allows monitoring of transcriptomic responses and selectively profiles cell-type-specific transcriptomes from mixed populations without cell dissociation and sorting. Additionally, POND-seq supports functional interrogation of RBP domains and residues involved in RNA association and enables scalable analysis of RBP variants, as demonstrated by a systematic assessment of disease-associated fragile X messenger ribonucleoprotein 1 (FMR1) mutations. Together, POND-seq provides a versatile and scalable platform for non-destructive and longitudinal analysis of cytoplasmic transcriptomes and RBP-associated RNAs.
PI3Kα is a potent oncogene that converts PIP2 to PIP3 at the plasma membrane upon activation by receptor tyrosine kinases and Ras. To understand the molecular mechanism of PI3Kα activation, we used cryo-electron microsco...PI3Kα is a potent oncogene that converts PIP2 to PIP3 at the plasma membrane upon activation by receptor tyrosine kinases and Ras. To understand the molecular mechanism of PI3Kα activation, we used cryo-electron microscopy to visualize the conformational states that underlie its transition to an active signaling complex. Here, we present structures of the PI3Kα/KRas complex embedded in lipid nanodiscs, revealing a rich ensemble of PI3Kα conformations that capture the progressive release of key inhibitory domains from the PI3Kα catalytic core. PIP2 triggers significant restructuring of active site regulatory motifs while an activating phosphopeptide induces dimerization of the PI3Kα/KRas complex through a p110α catalytic subunit-mediated interface that is sterically occluded in autoinhibited PI3Kα. In cells, dimeric PI3Kα amplifies Akt signaling in response to growth factor stimulation. Collectively, these structures map the conformational landscape of PI3Kα activation and reveal previously unexplored interfaces for potential therapeutic targeting.
The evolutionary arms race between bacteria and phages has driven the diversification of prokaryotic antiviral defense mechanisms, with nucleic acid degradation emerging as a central strategy. Here, we investigate a High...The evolutionary arms race between bacteria and phages has driven the diversification of prokaryotic antiviral defense mechanisms, with nucleic acid degradation emerging as a central strategy. Here, we investigate a Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domain-containing defense system from Escherichia coli that mediates RNase-dependent abortive infection. In contrast to canonical immune systems, where oligomerization triggers signaling, this system adopts a dodecameric autoinhibited architecture, with RNase activity unleashed upon oligomer dissociation. This activation mechanism is reminiscent of the dispersal of dandelion seeds, and we therefore term this defense system "Dandelion." We further identify the phage single-stranded DNA-binding (SSB) protein as a trigger for the Dandelion system, and phylogenetic analysis of SSB proteins uncovers the specificity underlying phage resistance. Our findings reveal a counterintuitive paradigm in bacterial immunity-oligomer disassembly as an activation switch, which challenges the long-standing dogma that protein oligomerization activates immune signaling.
The ∼2,500 multipass membrane proteins encoded in the human genome are constructed mostly or entirely of transmembrane domain (TMD) pairs: exceptionally common biosynthetic units comprised of two TMDs separated by a shor...The ∼2,500 multipass membrane proteins encoded in the human genome are constructed mostly or entirely of transmembrane domain (TMD) pairs: exceptionally common biosynthetic units comprised of two TMDs separated by a short non-cytosolic loop. It has long been thought that each TMD of a pair sequentially enters the lipid bilayer through a lateral gate in the Sec61 protein translocation channel. Here, we show that TMD pairs can access multiple insertion routes and that most are completely impervious to small-molecule blockade of Sec61's lateral gate. Obligate use of Sec61 is seen only for exceptional cases where the translocated loop exceeds ∼60 amino acids. TMD pairs with shorter loops typically use either EMC or GEL, insertase complexes of the universally conserved Oxa1 superfamily. Our results suggest that, contrary to long-held Sec61-based models, the fundamental biosynthetic unit of nearly all multipass membrane proteins uses the Oxa1 family for insertion.
Activation-induced cytidine deaminase (AID) converts cytosines to uracils in actively transcribed switch regions to initiate the formation of DNA double-strand breaks required for immunoglobulin class switch recombinatio...Activation-induced cytidine deaminase (AID) converts cytosines to uracils in actively transcribed switch regions to initiate the formation of DNA double-strand breaks required for immunoglobulin class switch recombination (CSR). How AID targets switch regions remains a key unanswered question. Using multimodal live-cell single-molecule imaging of mouse B cells, we demonstrate that intronic switch regions promote robust transcription by enhancing polymerase engagement and persistent transcriptional bursts, resulting in the formation of a dynamic RNA hub consisting of numerous nascent switch transcripts tethered to the IgH locus. We further demonstrate that AID interacts with switch region RNA in vivo and that this interaction is required for recruitment of AID to the IgH locus. Together, our findings show that the RNA hub formed by nascent switch region transcripts may be a part of a "class switch recombination center" and drives the recruitment of AID to the IgH locus to initiate CSR.
Biomolecular condensates are emerging structures that organize cell biochemistry. RNA-protein (RNP) condensates have raised huge interest in the field of RNA biology due to their potential to impact gene expression. Alth...Biomolecular condensates are emerging structures that organize cell biochemistry. RNA-protein (RNP) condensates have raised huge interest in the field of RNA biology due to their potential to impact gene expression. Although RNP condensate biophysical properties and assembly mechanisms have been extensively studied, leading to major breakthroughs, their contribution to biological processes remains debated. In this perspective, we review the current knowledge on the functions of cytoplasmic RNP condensates in mRNA regulation. Particularly, we highlight recent technological and conceptual advances that revealed the unexpected function of RNP condensates in mRNA translation. We discuss the mechanisms and biophysical bases that reconcile RNP condensate dual function in translational repression and activation. We propose emerging future directions to further address translation at RNP condensates and decode their functional compartmentalization linked to their biophysical properties. We also highlight the importance of this new function of condensates in translation for improved RNA-based therapeutics.
How the twin-arginine translocase (Tat) system transports fully folded substrate proteins across cellular membranes without disrupting membrane integrity has been a fundamental question in cell biology for decades. The T...How the twin-arginine translocase (Tat) system transports fully folded substrate proteins across cellular membranes without disrupting membrane integrity has been a fundamental question in cell biology for decades. The Tat system, found in prokaryotes and plant organelles, recognizes a cargo signal peptide via a conserved twin-arginine motif. The multi-subunit Tat complex facilitates the proton-motive-force-dependent translocation process, yet its overall architecture has remained unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure of the Escherichia coli (E. coli) trimeric TatB₃C₃ complex with bound substrate SufI, assembled in vivo. The complex adopts an unusual, wide-open, bowl-shaped architecture with a polar inner cavity. Unexpectedly, the cargo is engaged in a dual-contact mode: while the signal peptide binds inside one TatBC unit, the folded domain docks tightly onto an adjacent unit, possibly performing a proofreading function. This structure provides a mechanistic framework for substrate engagement and suggests the direct involvement of the entire Tat complex in substrate translocation.
Tiwari D, Sawada K, Dalal A
… +26 more, Mishra S, Li XX, Dent JC, Kim K, Yadav MK, Roy N, Ganguly M, Banerjee N, Stepniewski TM, Ahn D, Yamaguchi K, Oshima HS, Hashimoto K, Fung JN, Lerskiatiphanich T, Cui CS, Lee JD, Selent J, Inoue A, Clark RJ, Chung KY, Banerjee R, Sano FK, Woodruff TM, Nureki O, Shukla AK
The conceptual framework of biased agonism has greatly impacted our understanding of G-protein-coupled receptor (GPCR) signaling, regulatory paradigms, and drug discovery efforts. Here, we present fundamental molecular a...The conceptual framework of biased agonism has greatly impacted our understanding of G-protein-coupled receptor (GPCR) signaling, regulatory paradigms, and drug discovery efforts. Here, we present fundamental molecular and structural insights into intrinsic bias encoded at the human and mouse complement anaphylatoxin C5a receptors, namely C5aR1 and C5aR2. We discover that a naturally occurring version of C5a, i.e., C5a, exhibits a robust G-protein-coupling bias at C5aR1 with attenuated β-arrestin (βarr) recruitment, which originates from a distinct conformation of TM7 and helix 8 in the receptor, leading to inefficient GRK recruitment and phosphorylation. We also determine a series of cryo-electron microscopy (cryo-EM) structures of C5aR2, a naturally encoded βarr-biased receptor, which uncover key differences in anaphylatoxin recognition by C5aR2 relative to C5aR1. These structural snapshots also uncover a shallower cytoplasmic pocket in C5aR2 with a hydrophobic interior, which is likely incompatible with efficient G-protein coupling, leading to intrinsic bias. Our findings illuminate the molecular basis of naturally encoded signaling bias at GPCRs, with direct implications for therapeutic design.
Adaptation to fluctuating nutrient supply is essential for organismal survival, but how human cells monitor the abundance of many critical nutrients remains undefined. Characterizing the conditional degradation of CDO1,...Adaptation to fluctuating nutrient supply is essential for organismal survival, but how human cells monitor the abundance of many critical nutrients remains undefined. Characterizing the conditional degradation of CDO1, the critical enzyme responsible for cysteine catabolism, here we identify a Cullin-RING E3 ligase complex defined by the substrate adaptor LRRC58 that is sensitive to cysteine abundance. When cysteine is replete, LRRC58 activity is restrained through ubiquitination and proteasomal degradation. Upon cysteine deprivation, LRRC58 is stabilized to permit CDO1 degradation. Through saturation mutagenesis stability profiling, we systematically validate a structural model of the CDO1-LRRC58 interaction and identify residues at the LRRC58 C terminus required for cysteine-dependent instability. CDO1 degradation prevents ferroptotic cell death upon cysteine scarcity, and CDO1 mutations causing neurodevelopmental defects in humans encode dominant-active proteins refractory to LRRC58 recognition. Altogether, these data reveal the CDO1-LRRC58 axis as a critical regulator of cysteine homeostasis that safeguards neural development.