RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

Using the structural diversity of RNA: protein interfaces to selectively target RNA with small molecules in cells: methods and perspectives.

In recent years, RNA has gained traction both as a therapeutic molecule and as a therapeutic target in several human pathologies. In this review, we consider the approach of targeting RNA using small molecules for both research and therapeutic purposes. Given the primary challenge presented by the low structural diversity of RNA, we discuss the potential for targeting RNA: protein interactions to enhance the structural and sequence specificity of drug candidates. We review available tools and inherent challenges in this approach, ranging from adapted bioinformatics tools to in vitro and cellular high-throughput screening and functional analysis. We further consider two critical steps in targeting RNA/protein interactions: first, the integration of in silico and structural analyses to improve the efficacy of molecules by identifying scaffolds with high affinity, and second, increasing the likelihood of identifying on-target compounds in cells through a combination of high-throughput approaches and functional assays. We anticipate that the development of a new class of molecules targeting RNA: protein interactions to prevent physio-pathological mechanisms could significantly expand the arsenal of effective therapeutic compounds.

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation.

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors.

RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.

p53 and HuR combinatorially control the biphasic dynamics of microRNA-125b in response to genotoxic stress.

Post-transcriptional regulation of p53, by the microRNA miR-125b and the RNA-binding protein HuR, controls p53 expression under genotoxic stress. p53 mRNA translation is repressed by miR-125b, tightly regulating its basal level of expression. The repression is relieved upon DNA damage by a decrease in miR-125b level, contributing to pulsatile expression of p53. The pulse of p53, as also of HuR, in response to UV irradiation coincides with a time-dependent biphasic change in miR-125b level. We show that the cause for the decrease in miR-125b level immediately post DNA-damage is enhanced exosomal export mediated by HuR. The subsequent increase in miR-125b level is due to p53-mediated transcriptional upregulation and enhanced processing, demonstrating miR-125b as a transcriptional and processing target of p53. p53 activates the transcription of primary miR-125b RNA from a cryptic promoter in response to UV irradiation. Together, these regulatory processes constitute reciprocal feedback loops that determine the biphasic change in miR-125b level, ultimately contributing to the fine-tuned temporal regulation of p53 expression in response to genotoxic stress.

PARP1 Activation Controls Stress Granule Assembly after Oxidative Stress and DNA Damage.

DNA damage causes PARP1 activation in the nucleus to set up the machinery responsible for the DNA damage response. Here, we report that, in contrast to cytoplasmic PARPs, the synthesis of poly(ADP-ribose) by PARP1 opposes the formation of cytoplasmic mRNA-rich granules after arsenite exposure by reducing polysome dissociation. However, when mRNA-rich granules are pre-formed, whether in the cytoplasm or nucleus, PARP1 activation positively regulates their assembly, though without additional recruitment of poly(ADP-ribose) in stress granules. In addition, PARP1 promotes the formation of TDP-43- and FUS-rich granules in the cytoplasm, two RNA-binding proteins which form neuronal cytoplasmic inclusions observed in certain neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Together, the results therefore reveal a dual role of PARP1 activation which, on the one hand, prevents the early stage of stress granule assembly and, on the other hand, enables the persistence of cytoplasmic mRNA-rich granules in cells which may be detrimental in aging neurons.

FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins.

Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS-nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS's affinity for a nucleic acid polymer. Some proteins-which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates-disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.

The C-Terminal Domain of Y-Box Binding Protein 1 Exhibits Structure-Specific Binding to Poly(ADP-Ribose), Which Regulates PARP1 Activity.

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in the regulation of gene expression. Recent studies showed that in addition to its role in the RNA and DNA metabolism, YB-1 is involved in the regulation of PARP1 activity, which catalyzes poly(ADP-ribose) [PAR] synthesis under genotoxic stress through auto-poly(ADP-ribosyl)ation or protein trans-poly(ADP-ribosyl)ation. Nonetheless, the exact mechanism by which YB-1 regulates PAR synthesis remains to be determined. YB-1 contains a disordered Ala/Pro-rich N-terminal domain, a cold shock domain, and an intrinsically disordered C-terminal domain (CTD) carrying four clusters of positively charged amino acid residues. Here, we examined the functional role of the disordered CTD of YB-1 in PAR binding and in the regulation of PARP1-driven PAR synthesis in vitro. We demonstrated that the rate of PARP1-dependent synthesis of PAR is higher in the presence of YB-1 and is tightly controlled by the interaction between YB-1 CTD and PAR. Moreover, YB-1 acts as an effective cofactor in the PAR synthesis catalyzed by the PARP1 point mutants that generate various PAR polymeric structures, namely, short hypo- or hyperbranched polymers. We showed that either a decrease in chain length or an increase in branching frequency of PAR affect its binding affinity for YB-1 and YB-1-mediated stimulation of PARP1 enzymatic activity. These results provide important insight into the mechanism underlying the regulation of PARP1 activity by PAR-binding proteins containing disordered regions with clusters of positively charged amino acid residues, suggesting that YB-1 CTD-like domains may be considered PAR "readers" just as other known PAR-binding modules.

YB-1 Structure/Function Relationship in the Packaging of mRNPs and Consequences for Translation Regulation and Stress Granule Assembly in Cells.

From their synthesis in the nucleus to their degradation in the cytoplasm, all mRNAs have the same objective, which is to translate the DNA-stored genetic information into functional proteins at the proper time and location. To this end, many proteins are generally associated with mRNAs as soon as transcription takes place in the nucleus to organize spatiotemporal regulation of the gene expression in cells. Here we reviewed how YB-1 (YBX1 gene), one of the major mRNA-binding proteins in the cytoplasm, packaged mRNAs into either compact or extended linear nucleoprotein mRNPs. Interestingly the structural plasticity of mRNPs coordinated by YB-1 could provide means for the contextual regulation of mRNA translation. Posttranslational modification of YB-1, notably in the long unstructured YB-1 C-terminal domain (CTD), and/or the protein partners of YB-1 may play a key role in activation/inactivation of mRNPs in the cells notably in response to cellular stress.

Identification of a small molecule splicing inhibitor targeting UHM domains.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

The cooperative binding of TDP-43 to GU-rich RNA repeats antagonizes TDP-43 aggregation.

TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

Molecular dynamics of the histamine H3 membrane receptor reveals different mechanisms of GPCR signal transduction.

In this work, we studied the mechanisms of classical activation and inactivation of signal transduction by the histamine H3 receptor, a 7-helix transmembrane bundle G-Protein Coupled Receptor through long-time-scale atomistic molecular dynamics simulations of the receptor embedded in a hydrated double layer of dipalmitoyl phosphatidyl choline, a zwitterionic polysaturated ordered lipid. Three systems were prepared: the apo receptor, representing the constitutively active receptor; and two holo-receptors-the receptor coupled to the antagonist/inverse agonist ciproxifan, representing the inactive state of the receptor, and the receptor coupled to the endogenous agonist histamine and representing the active state of the receptor. An extensive analysis of the simulation showed that the three states of H3R present significant structural and dynamical differences as well as a complex behavior given that the measured properties interact in multiple and interdependent ways. In addition, the simulations described an unexpected escape of histamine from the orthosteric binding site, in agreement with the experimental modest affinities and rapid off-rates of agonists.

Regulation of Poly(ADP-Ribose) Polymerase 1 Activity by Y-Box-Binding Protein 1.

Y-box-binding protein 1 (YB-1) is a multifunctional positively charged protein that interacts with DNA or RNA and poly(ADP-ribose) (PAR). YB-1 is poly(ADP-ribosyl)ated and stimulates poly(ADP-ribose) polymerase 1 (PARP1) activity. Here, we studied the mechanism of YB-1-dependent PAR synthesis by PARP1 in vitro using biochemical and atomic force microscopy assays. PAR synthesis activity of PARP1 is known to be facilitated by co-factors such as Mg2+. However, in contrast to an Mg2+-dependent reaction, the activation of PARP1 by YB-1 is accompanied by overall up-regulation of protein PARylation and shortening of the PAR polymer. Therefore, YB-1 and cation co-factors stimulated PAR synthesis in divergent ways. PARP1 autoPARylation in the presence of YB-1 as well as trans-PARylation of YB-1 are greatly affected by the type of damaged DNA, suggesting that PARP1 activation depends on the formation of a PARP1-YB-1-DNA ternary complex. An unstructured C-terminal part of YB-1 involved in an interaction with PAR behaves similarly to full-length YB-1, indicating that both DNA and PAR binding are involved in the stimulation of PARP1 activity by YB-1. Thus, YB-1 is likely linked to the regulation of PARylation events in cells via an interaction with PAR and damaged DNA.

Fused in Sarcoma (FUS) in DNA Repair: Tango with Poly(ADP-ribose) Polymerase 1 and Compartmentalisation of Damaged DNA.

The fused in sarcoma (FUS) protein combines prion-like properties with a multifunctional DNA/RNA-binding domain and has functions spanning the regulation of RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and translation. In addition to its roles in RNA metabolism, FUS is implicated in the maintenance of DNA integrity. In this review, we examine the participation of FUS in major DNA repair pathways, focusing on DNA repair associated with poly(ADP-ribosyl)ation events and on how the interaction of FUS with poly(ADP-ribose) may orchestrate transient compartmentalisation of DNA strand breaks. Unravelling how prion-like RNA-binding proteins control DNA repair pathways will deepen our understanding of the pathogenesis of some neurological diseases and cancer as well as provide the basis for the development of relevant innovative therapeutic technologies. This knowledge may also extend the range of applications of poly(ADP-ribose) polymerase inhibitors to the treatment of neurodegenerative diseases related to RNA-binding proteins in the cell, e.g., amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

U2AF65 assemblies drive sequence-specific splice site recognition.

The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates.

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD+ as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.