Molecular Cell

Neuronal activity causes topoisomerase II{beta} (TOP2B) to form DNA double strand breaks (DSBs) within the promoters of key early response genes (ERGs), such as Fos and Npas4. TOP2B-mediated DSBs facilitate rapid ERG transcription, yet how this occurs remains unclear. Here, using chromosome conformation capture methods (3C and 4C-seq), we report that DSB formation within the promoters of Fos and Npas4 is sufficient to emulate contact profiles observed at these regions following neuronal stimulation, including their elevated interactions with cognate enhancers. Furthermore, despite their purported risk, repeated DSB cycles within ERG promoters progressively potentiated ERG induction in both mouse cortical neurons and HEK293T cells, evoking the effects of transcriptional memory. Potentiated ERG inducibility following recurrent DSBs persisted through intervening cell cycles, occurred even when DNA repair was likely mutagenic, and was associated with a substantial loss of cis chromosome interactions and an increase in trans interactions with ERG promoters. Together, these results reveal how single and recurrent TOP2B-mediated DSBs could affect stimulus-dependent transcription patterns by affecting chromatin dynamics at ERG promoters.

Replication disrupts chromatin organization. Thus, the rapid resetting of nucleosome positioning is essential to maintain faithful gene expression. The initial step of this reconfiguration occurs at Nucleosome-Depleted Regions (NDRs). While studies have elucidated the role of Transcription Factors (TFs) and Chromatin Remodelers (CRs) in vitro or in maintaining NDRs in vivo, none has addressed their in vivo function shortly after replication. Through purification of nascent chromatin in yeast, we dissected the choreography of events governing the proper positioning of the -1/+1 nucleosomes flanking promoter NDRs. Our findings reveal that CRs are the primary contributors of -1/+1 repositioning post-replication, with RSC acting upstream of INO80. Surprisingly, while Reb1 and Abf1 TFs are not essential for NDR resetting, they are required for NDR maintenance via the promotion of H3 acetylations. Altogether, we propose a two-step model for NDR resetting in S. cerevisiae: first, CRs alone reset promoter NDRs after replication, while a combination of TFs and CRs is required for subsequent maintenance. TeaserRSC acts upstream of INO80 for NDR re-establishment after replication followed by a combined action of CRs and TFs for NDR maintenance.

Promoter-proximal RNA Polymerase II (RNAPII) pausing and the processivity are controlled by distinct modules of the Integrator complex, which together fine-tune transcription and protect against the accumulation of defective RNAPII complexes. Compromised activity of individual Integrator modules has been linked to human disease including cancer and developmental disorders, caused by defective transcription of protein-coding or small-nuclear RNAs. Despite extensive characterisation of the Integrator complex both genetically and structurally, the role of smallest member of the complex, INTS12, has remained enigmatic. Here, we uncover that INTS12 loss acts to stabilise the association between NELF and Integrator via its PHD domain and N-terminus, respectively, thus safeguarding against the release of defective RNAPII complexes. Acute degradation of INTS12 results in the selective dissociation of Integrator from the NELF-RNAPII complex which subsequently convert to their canonical paused form from which they can be released by CDK9. In the absence of INTS12 excess release of defective RNAPII via P-TEFb/SEC, loss of the ARMC5 salvage pathway and deletion of the catalytic and core Integrator subunits is toxic to cells. These findings demonstrate that there is interconversion between canonical paused RNAPII and paused-Integrator, and highlight the critical interplay between these processes and P-TEFb mediated pause-release to ensure that only transcription competent complexes are released into elongation. O_LIINTS12 degradation confers CDK9 inhibitor resistance and triggers cellular stress through a phosphatase module-independent mechanism. C_LIO_LIINTS12 stabilizes the Integrator-NELF complex through its N-terminus and PHD domain. C_LIO_LIAcute INTS12 degradation promotes aberrant release of promoter-proximal RNA polymerase II complexes. C_LIO_LIRNA polymerase II complexes released upon INTS12 loss exhibit defective elongation and reduced processivity. C_LIO_LIINTS12 loss removes Integrator from RNAPII resulting in aberrant paused-state from which it can be released by CDK9. C_LIO_LIExcess CDK9 activity and ARMC5 loss are synthetically lethal with INTS12 deficiency. C_LI

Modifications of histones are intricately linked with the regulation of gene expression, with demonstrated roles in various physiological processes and disease pathogenesis. Methylation of histone H3 lysine 4 (H3K4), implemented by the COMPASS family, is enriched at promoters and associated cis-regulatory elements, with H3K4 trimethylation (H3K4me3) considered a hallmark of active gene promoters. However, the relative roles of deposition and removal of H3K4 methylation, as well as the extent to which these events contribute to transcriptional regulation have so far remained unclear. Here, through rapid depletion of the transcription regulator SPT5 or either of two shared subunits of COMPASS family members, we reveal a dynamic turnover of H3K4me3 mediated by the KDM5 family of histone demethylases. Loss of H3K4me3 following COMPASS disruption does not impair the recruitment of TFIID and initiating RNA polymerase II (Pol II). Instead, H3K4me3 loss leads to reductions in the paused form of Pol II on chromatin while inducing the relative enrichment of the Integrator-PP2A (INTAC) termination complex, leading to reduced levels of elongating polymerases, thus revealing how H3K4me3 dynamics can regulate Pol II pausing to sustain or attenuate transcription.

RB1 (retinoblastoma) members control the G1/S commitment as transcriptional repressors in eukaryotic cells. Here we uncover that an extra copy of RB1 equivalent (WHI7 or WHI5) is sufficient to bypass the indispensability of the central genomic checkpoint kinases Mec1ATR-Rad53CHK1 in Saccharomyces cerevisiae. Mec1-Rad53 directly phosphorylate Whi7/5, antagonizing their nuclear export or protein turnover upon replication stress. Through in vitro reconstitution, we show that Whi7 C-terminus directly binds and hinders S-CDK-Cks1 from processively phosphorylating Sic1. By microfluidic single-cell real-time quantitative imaging, we demonstrate that both Whi7 and Whi5 are required to flatten the degradation curve of the major S-CDK inhibitor Sic1 in vivo. These findings reveal an eclipsed transcription-independent role of Whi7 homologs, which is highlighted by genome integrity checkpoints to hold the G1/S transition instantly as a rapid response to unforeseeable replication threats. Key pointsO_LIWhi7 overexpression bypasses the essential function of Mec1 and Rad53 in a transcription-independent way. C_LIO_LIWhi7 is stabilized by checkpoint-mediated phosphorylation. C_LIO_LIWhi7 binds and hinders S-CDK-Cks1 from multi-phosphorylation of Sci1, thereby prolonging Sic1 degradation and G1/S transition. C_LI

RNA polymerase III (Pol III) produces noncoding RNAs involved in diverse cellular activities, including translation (tRNA, 5S rRNA, 7SL RNA), RNA processing (U6 snRNA, RPPH1, RMRP), and transcription regulation (7SK snRNA). In this way, Pol III activity must be broadly coupled with cellular demands for protein accumulation and growth, increasing in response to nutrient availability and decreasing during differentiation and exit from proliferation. However, the currently established mechanisms of Pol III regulation remain relatively limited, due in part to the few Pol III-centered protein-protein interaction (PPI) studies performed to date. To address this gap, we first investigated PPIs shared by multiple Pol III subunits to understand the macromolecular interactome of Pol III, with special attention directed at potential regulatory candidates. Our proteomic survey uncovers interactions between Pol III and the NuRD (Nucleosome Remodeling and Deacetylase) complex. Taken further, we show that NuRD localizes to active Pol III-transcribed genes and that its recruitment is Pol III-dependent but nonrandom, with peak occupancy and regulatory hallmarks converging on tRNA gene clusters associated with notably high expression levels. Inhibiting NuRD-associated histone deacetylase function reduces Pol III transcription at these sites, suggesting NuRD restricts Pol III and thereby modulates the global dynamic range of Pol III-derived RNA species. These findings are congruent with the transcriptionally repressive nature of NuRD and bring-to-light a new regulatory mechanism that may couple signaling events and changes in metabolic needs with the dynamic availability of specific tRNA pools.

Human quinone reductase 2 (QR2, NQO2) is a cytosolic flavoprotein involved in cell physiology and metabolism, and implicated in several diseases. However, the mechanisms that govern its oligomeric assembly and diverse functional outcomes remain incompletely understood. Here, we employ native mass spectrometry to directly resolve the dynamic oligomeric landscape of recombinant human QR2 expressed in Escherichia coli, preserving non-covalent interactions and enabling analysis of assembly behavior under native conditions. QR2 is predominantly observed as a dimer stabilized by multiple non-covalently bound ligands, giving rise to discrete species. Top-down native mass spectrometry reveals a single intact proteoform, excluding covalent modification or covalently bound flavins as drivers of oligomerization. Binding of flavin adenine dinucleotide (FAD) robustly stabilizes the dimer, while unexpectedly, flavin mononucleotide (FMN) also promotes dimer formation. As FMN and FAD differ structurally by the presence of an adenine dinucleotide moiety, we hypothesized that purine nucleotide binding itself may modulate QR2 assembly. Consistent with this, we identify a new concentration-dependent effect of guanosine-triphosphate (GTP) on QR2 dimerization. Functional reductase assays show that flavin-stabilized dimers exhibit the highest catalytic activity, whereas GTP-induced dimers retain reduced activity. Binding of the inhibitor YB537 abolishes activity despite promoting dimer formation. Together, these findings reveal a ligand-dependent structural plasticity in QR2 oligomerization that is decoupled from reductase function, suggesting that QR2 dimerization serves a wider regulatory role beyond simply supporting reductase catalysis.

Organisms with smaller genomes often perform multiple functions using one multi-subunit protein complex. The S. cerevisiae Silent Information Regulator complex (SIRc) carries out all of the core functions of heterochromatin. SIR complexes first drive the initiation and spreading of histone deacetylation in an iterative manner. Subsequently, the same complexes are incorporated stably with nucleosomes, driving compaction and repression of the underlying chromatin domain. These two distinct functions of SIRc have each been characterized in much detail, but the mechanism by which the dynamic spreading state switches to stable compaction is not well-understood. This incomplete knowledge of potential intra-complex communication is partly due to a lack of structural information of the complex as a whole; only structures of fragments have been determined to date. Using cross-linking mass spectrometry in solution, we identified a novel inter-subunit interaction that physically connects the two states of SIRc. The Sir2 deacetylase makes direct interactions with the scaffolding subunit Sir4 through its coiled-coil domain, which also interacts with the Sir3 compaction/repression subunit. Within the hub of interactions are conserved residues in Sir2 that can sense deacetylation state, as well as amino acids that likely diverged and co-evolved to interact with Sir4, promoting species-specific functions. Mutation of this interaction hub disrupts heterochromatic repression, potentially by disrupting a conserved mechanism that communicates completion of deacetylation to switch to compaction. Our work highlights how a single multi-functional chromatin regulatory complex can stage a step-wise mechanism that requires a major transition in activities to achieve epigenetic gene repression.

In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression post pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutS and enhances the chromatin-association of MutL, thus enabling efficient mismatch repair. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S-phase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/443400v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@bfdaa8org.highwire.dtl.DTLVardef@1ac791dorg.highwire.dtl.DTLVardef@436d1corg.highwire.dtl.DTLVardef@b07689_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHuman Orc6 is dispensable for G1 licensing, but required for S-phase progression C_LIO_LIHuman Orc6 at the replication fork is an accessory factor for MMR complex C_LIO_LIDepletion of hOrc6 sensitizes cells to DNA damage and impairs ATR activation C_LIO_LIHuman Orc6 regulates MMR complex assembly and activity C_LI

NIPBL promotes chromatin loop extrusion by the cohesin complex until it stalls at convergently oriented CTCF sites, leading to the formation of structural loops. However, to what extent loop extrusion contributes to the establishment vs maintenance of cis-regulatory element (CRE) connectivity is poorly understood. Here, we explored the de novo establishment of chromatin folding patterns at the mitosis-to-G1-phase transition upon acute NIPBL loss. NIPBL depletion primarily impaired the formation of cohesin-mediated structural loops with NIPBL dependence being proportional to loop length. In contrast, the majority of CRE loops were established independently of loop extrusion regardless of length. However, NIPBL depletion slowed the re-formation of CRE loops with weak enhancers. Transcription of genes at NIPBL-independent loop anchors was activated normally in the absence of NIPBL. In sum, establishment of most regulatory contacts and gene transcription following mitotic exit is independent of loop extrusion.

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates -- splicing, export, localisation, decay, stability, and translation -- to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in the m6A pathway. MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A-containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3{beta} mRNA through m6A that stabilises the {beta}-CATENIN expression of a WNT/{beta}-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mouse embryonic stem cells (mESCs) state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

Efficient DNA double strand break (DSB) repair by homologous recombination (HR), as orchestrated by histone and non-histone proteins, is critical to genome stability, replication, transcription, and cancer avoidance. Here we report that Heterochromatin Protein1 beta (HP1{beta}) acts as a key component of the HR DNA resection step by regulating BRCA1 enrichment at DNA damage sites, a function largely dependent on the HP1{beta} chromo shadow domain (CSD). HP1{beta} itself is enriched at DSBs within gene-rich regions through a CSD interaction with Chromatin Assembly Factor 1 (CAF1) and HP1 {beta} depletion impairs subsequent BRCA1 enrichment. An added interaction of the HP1 {beta} CSD with the Polycomb Repressor Complex 1 ubiquitinase component RING1A facilitates BRCA1 recruitment by increasing H2A lysine 118-119 ubiquitination, a marker for BRCA1 recruitment. Our findings reveal that HP1{beta} interactions, mediated through its CSD with RING1A, promote H2A ubiquitination and facilitate BRCA1 recruitment at DNA damage sites, a critical step in DSB repair by the HR pathway. These collective results unveil how HP1{beta} is recruited to DSBs in gene-rich regions and how HP1{beta} subsequently promotes BRCA1 recruitment to further HR DNA damage repair by stimulating CtIP-dependent resection.

The Elg1 Replication Factor C-like complex (Elg1-RLC) that functions as a PCNA unloader, is known to be involved in multiple DNA replication/repair-related activities from yeast to humans. By exploiting disassembly-prone PCNA mutants, we reveal that Elg1-RLC uses its PCNA unloading activity to counter the DNA-alkylating agent methyl-methanesulfonate (MMS)-mediated slow progression of replication forks. Despite having a largely functional DNA Damage Response (DDR), the viability loss of elg1{Delta}-DDR double mutants, in the presence of MMS, matches that of mec1{Delta} and rad53{Delta} cells, deficient for the central checkpoint kinases. This suggests that elg1{Delta}-DDR double mutants experience replication fork collapse when exposed to MMS. Indeed, in response to MMS, accumulation of Rad52 foci in the replicative elg1{Delta}-DDR cells supports this possibility. However, the failure of rescuing elg1{Delta}-DDR mutants by elevating dNTP levels (by deleting the ribonucleotide reductase SML1) eliminates the possibility of a Rad53-regulated dNTP shortage-mediated fork collapse. Thus, we propose a S-phase checkpoint regulatory role of Elg1-RLC that works through a noncanonical pathway parallel to the canonical one. Collectively, our findings suggest a model in which Elg1-RLC, by timely unloading chromatin-bound PCNA from the damaged/stalled forks, coordinates the DDR pathways to safeguard the integrity of replication forks under replication stress.

Proteolytic processing is a critical regulatory mechanism in eukaryotic cells, yet the molecular identities and mechanisms underlying these events often remain elusive. Silencing Defective 2 (SDE2) is an essential human protein involved in multiple aspects of genome regulation, including DNA repair, ribosome biogenesis, and mRNA splicing. SDE2 is expressed with an N-terminal ubiquitin-like domain (SDE2UBL) which must be proteolytically cleaved to release the functional C-terminal domain (SDE2CT). This cleavage not only activates SDE2CT but also marks it for subsequent degradation, highlighting the importance of this tightly regulated processing. Despite the crucial role of this cleavage event, the human protease responsible has remained unknown. Here, we identify that the deubiquitinating enzyme, ubiquitin-specific protease 5 (USP5), catalyses the cleavage of SDE2. Using an integrated workflow combining biochemical assays, proteomic profiling, and mass spectrometry, we demonstrate that USP5 selectively processes SDE2 in vitro and in cell. To validate the specificity of this interaction, we engineered SDE2UBL into an activity-based probe, and developed a cellular reporter assay, both of which confirmed USP5 as the primary effector. Biophysical analysis further revealed that SDE2UBL binds to USP5 with similar characteristics to ubiquitin, albeit with reduced affinity, supporting a mechanism of substrate mimicry. Together, these findings uncover a novel regulatory axis for SDE2 function, highlighting an underappreciated role for DUBs in regulating protein maturation events. They also establish a versatile approach for identifying and validating substrate-protease interactions with broader implications for the study of post-translational regulation.

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

Stress granules (SGs) are dynamic, membrane-less assemblies that form in the cytoplasm in response to cellular stress. The ordered recruitment of proteins into SGs is fundamental to condensate composition and function, yet the molecular determinants of this ordered client recruitment remain incompletely understood. Using proximity photo-crosslinking proteomics, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a TIA1-proximal protein preferentially enriched in SGs under arsenite stress. Knockdown of hnRNPA2B1 preferentially delayed TIA1 enrichment in G3BP1-marked SGs at 20 min without affecting G3BP1 or the overall SG-positive cell fraction, and this phenotype showed directional rescue upon re-expression. In vitro droplet reconstitution assays with purified proteins revealed that hnRNPA2B1 and RNA cooperatively increased TIA1 incorporation capacity into G3BP1 condensates, an effect not attributable to changes in droplet size. Kinetic fitting identified hnRNPA2B1 + RNA as uniquely increasing the plateau amplitude of TIA1 recruitment (Cohens d = 1.62 versus RNA-alone condition). Coarse-grained simulations support an inside-out assembly model in which hnRNPA2B1 stabilizes the condensate core through homotypic interactions while RNA-bound TIA1 accumulates at the periphery. Together, these findings identify hnRNPA2B1 as a capacity-determining modulator of early TIA1 recruitment and provide a framework for understanding ordered protein assembly within stress granules.

ARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA Induced Silencing Complexes (RISCs). In the RNA-bound state, AGO proteins are stable while RNA-free AGOs turn over rapidly. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we show that a confined, linear region in Arabidopsis AGO1, the N-coil, is accessible to antibodies preferentially in the RNA-free state of AGO1. Reanalysis of hydrogen-deuterium exchange data on human Ago2 indicates similar structural flexibility of the N-coil depending on small RNA binding. Unloaded Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 via direct contact to specific amino acid residues in the N-coil, and mutation of residues required for ATI1 interaction reduces the degradation rate of unloaded AGO1 in vivo. These results provide insight into the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins.

Epigenetic regulation is tightly linked to cellular metabolism through chromatin-modifying enzymes that depend on central metabolites as co-substrates. Methionine is an essential amino acid that is directly converted by methionine adenosyltransferase 2A (MAT2A) into S-adenosylmethionine (SAM), the universal methyl donor required for histone and DNA methylation. Although methionine restriction/depletion can alter the chromatin methylation landscape and improve physiological outcomes in diverse biological systems, it remains unclear whether these effects arise from loss of methionine itself or from secondary depletion of SAM. Here, we show that methionine depletion induces nuclear accumulation of MAT2A together with redistribution of H3K9 methylation, derepression of transposable elements, activation of stress-response pathways, and broad transcriptional reprogramming. Surprisingly, pharmacologic inhibition reduced intracellular SAM to levels comparable to methionine depletion but failed to reproduce these major epigenetic or transcriptional responses. Furthermore, depletion of the SAM-sensor SAMTOR and inhibition of KDM4 histone demethylases did not prevent methionine-dependent chromatin remodeling, indicating that canonical SAM-sensing pathways are not required for this adaptation. Instead, methionine depletion uniquely induced innate immune and integrated stress-response programs consistent with a viral mimicry-like state. These findings demonstrate that methionine availability, rather than SAM abundance, functions as a primary metabolic signal regulating epigenetic adaptation to nutrient stress. Our data support a model in which methionine is sensed independently of SAM abundance and acts upstream of stress signaling pathways that secondarily remodel chromatin.

Mutations in, or deficiency of, FMRP is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently uncovered a genome protective role of FMRP. We reported that FXS patient-derived cells lacking FMRP sustain higher level of DNA double-strand breaks than normal cells, a phenotype further exacerbated by DNA replication stress. The stress-induced DSBs occur at sequences prone to form R-loops, which are co-transcriptional RNA:DNA hybrids that have been associated with genome instability. Concordantly, we showed that FXS cells accumulate R-loops under replication stress. Moreover, expression of FMRP and not a mutant deficient in binding nucleic acids and known to cause FXS, FMRPI304N, reduced R-loop-associated DSBs. These observations demonstrated that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. Here, we explore the mechanism through which FMRP prevents R-loop accumulation in an isogenically controlled CRISPR KO of FMR1 (gene encoding for FMRP) in HEK293T cells. We demonstrate for the first time that FMRP directly binds R-loops. We show that FMRP interacts with DHX9, an RNA helicase that unwinds both double strand RNA and RNA:DNA hybrids and regulates R-loop formation through modulating these activities. This interaction is reduced with FMRPI304N, suggesting that FMRP regulation of R-loop is mediated through DHX9. Interestingly, we show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids. Moreover, DHX9 binds chromatin containing R-loops more efficiently in the absence of a functional FMRP. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where FMRP prevents R-loop formation by suppressing DHX9. Our study sheds new light on our understanding of the genome functions of FMRP.

Eukaryotic translation initiation factor (eIF) 4A -- a DEAD-box RNA-binding protein -- plays an essential role in translation initiation. Two mammalian eIF4A paralogs, eIF4A1 and eIF4A2, have been assumed to be redundant because of their high homology, and the difference in their functions has been poorly understood. Here, we show that eIF4A1, but not eIF4A2, enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA-immunoprecipitation sequencing (RIP-Seq) of the two eIF4A paralogs revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTOR inhibition. This biased interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that the deletion of EIF4A1, but not EIF4A2, rendered the translation of TOP mRNAs resistant to mTOR inactivation. Moreover, eIF4A1 enhances the affinity between TOP mRNAs and LARP1 and thus ensures stronger translation repression upon mTORC1 inhibition. Our data show that the distinct protein interactions of these highly homologous translation factor paralogs shape protein synthesis during mTORC1 inhibition and provide a unique example of the repressive role of a universal translation activator.