RNA Binding Sites Research Articles

Genome-Wide Identification of Stable RNA Secondary Structures Across Multiple Organisms Using Chemical Probing Data: Insights into Short Structural Motifs and RNA-Targeting Therapeutics.

Small molecules targeting specific RNA binding sites, including stable and transient RNA structures, are emerging as effective pharmacological approaches for modulating gene expression. However, little is understood about how stable RNA secondary structures are shared across organisms, an important factor in controlling drug selectivity. In this study, I provide an analytical pipeline named RNA Secondary Structure Finder (R2S-Finder) to discover short, stable RNA structural motifs for humans, Escherichia coli ( E. coli ), SARS-CoV-2, and Zika virus by leveraging existing in vivo and in vitro genome-wide chemical RNA-probing datasets. I found several common features across organisms. For example, apart from the well-documented tetraloops, AU-rich tetraloops are widely present in different organisms. I also found that the 5' untranslated region (UTR) contains a higher proportion of stable structures than the coding sequences in humans, SARS-CoV-2, and Zika virus. In general, stable structures predicted from in vitro (protein-free) and in vivo datasets are consistent in humans, E. coli , and SARS-CoV-2, indicating that most stable structure formation were driven by RNA folding alone, while a larger variation was found between in vitro and in vivo data with certain RNA types, such as human long intergenic non-coding RNAs (lincRNAs). Finally, I predicted stable three- and four-way RNA junctions that exist both in vivo and in vitro conditions, which can potentially serve as drug targets. All results of stable sequences, stem-loops, internal loops, bulges, and three- and four-way junctions have been collated in the R2S-Finder database ( https://github.com/JingxinWangLab/R2S-Finder ), which is coded in hyperlinked HTML pages and tabulated in CSV files.

Long-range conformational changes in the nucleotide-bound states of the DEAD-box helicase Vasa

DEAD-box helicases use ATP to unwind short double-stranded RNA (dsRNA). The helicase core consists of two discrete domains, termed RecA_N and RecA_C. The nucleotide binding site is harbored in RecA_N, while both RecA_N and RecA_C are involved in RNA recognition and ATP hydrolysis. In the absence of nucleotides or RNA, RecA_N and RecA_C do not interact (“open” form of the enzyme). In the presence of both RNA and ATP the two domains come together (“closed” form), building the composite RNA binding site and stimulating ATP hydrolysis. Because of the different roles and thermodynamic properties of the ADP-bound and ATP-bound states in the catalytic cycle, the conformations of DEAD-box helicases in complex with ATP and ADP are assumed to be different. However, the available crystal structures do not recapitulate these supposed differences and show identical conformations of DEAD-box helicases independent of the identity of the bound nucleotide. Here, we use NMR to demonstrate that the conformations of the ATP- and ADP-bound forms of the DEAD-box helicase Vasa are indeed different, contrary to the results from x-ray crystallography. These differences do not relate to the populations of the open and closed forms, but are intrinsic to the RecA_N domain. NMR chemical shift analysis reveals the regions of RecA_N where the average conformations of Vasa-ADP and Vasa-ATP are most different and indicates that these differences may contribute to modulating the affinity of the two nucleotide-bound complexes for RNA substrates.

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

PDNAPred: Interpretable prediction of protein-DNA binding sites based on pre-trained protein language models

Protein-DNA interactions play critical roles in various biological processes and are essential for drug discovery. However, traditional experimental methods are labor-intensive and unable to keep pace with the increasing volume of protein sequences, leading to a substantial number of proteins lacking DNA-binding annotations. Therefore, developing an efficient computational method to identify protein-DNA binding sites is crucial. Unfortunately, most existing computational methods rely on manually selected features or protein structure information, making these methods inapplicable to large-scale prediction tasks. In this study, we introduced PDNAPred, a sequence-based method that combines two pre-trained protein language models with a designed CNN-GRU network to identify DNA-binding sites. Additionally, to tackle the issue of imbalanced dataset samples, we employed focal loss. Our comprehensive experiments demonstrated that PDNAPred significantly improved the accuracy of DNA-binding site prediction, outperforming existing state-of-the-art sequence-based methods. Remarkably, PDNAPred also achieved results comparable to advanced structure-based methods. The designed CNN-GRU network enhances its capability to detect DNA-binding sites accurately. Furthermore, we validated the versatility of PDNAPred by training it on RNA-binding site datasets, showing its potential as a general framework for amino acid binding site prediction. Finally, we conducted model interpretability analysis to elucidate the reasons behind PDNAPred's outstanding performance.

Peptide-Based Nanoparticles Suppress Hepatic Inflammation via Blockage of Human Antigen R.

Human antigen R (HuR), which is a mRNA-binding protein that stabilizes and regulates mRNA translation, is found to have increased expression in inflammation, cancer and other diseases, making HuR to be a promising drug target. This study reports a peptide-based nanoparticle (NP) system exhibits potent anti-inflammatory activity to ameliorate acute liver injury via the ability of peptides to inhibit the mRNA binding site of HuR and block downstream signaling. Molecular modeling provided structural evidence indicating that the peptides interact with the RNA-binding site of HuR, mainly via hydrogen-bonding and hydrophobic interactions. These peptide-based NPs can act as nanocarriers to deliver peptides into cells to compete with the mRNA binding site of HuR, evidenced by the reduction of antibody recognition to the native protein and the exhibition of anti-inflammatory activity against activated macrophage cells, with no adverse effect in vitro and in vivo. In LPS/D-GalN-induced hepatic sepsis with high dosage of LPS/GalN, administration of the NPs significantly attenuated necrosis and HuR expression, resulting in the significant improvement of animal survival rate, suggesting their therapeutic potential for hepatic inflammation and a broad range of HuR-overexpressed diseases.

Capture of RNA-binding proteins across mouse tissues using HARD-AP

RNA-binding proteins (RBPs) modulate all aspects of RNA metabolism, but a comprehensive picture of RBP expression across tissues is lacking. Here, we describe our development of the method we call HARD-AP that robustly retrieves RBPs and tightly associated RNA regulatory complexes from cultured cells and fresh tissues. We successfully use HARD-AP to establish a comprehensive atlas of RBPs across mouse primary organs. We then systematically map RNA-binding sites of these RBPs using machine learning-based modeling. Notably, the modeling reveals that the LIM domain as an RNA-binding domain in many RBPs. We validate the LIM-domain-only protein Csrp1 as a tissue-dependent RNA binding protein. Taken together, HARD-AP is a powerful approach that can be used to identify RBPomes from any type of sample, allowing comprehensive and physiologically relevant networks of RNA-protein interactions.

Insights into the structure of NLR family member X1: Paving the way for innovative drug discovery

Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a negative regulator of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway, with a significant role in the context of inflammation. Altered expression of NLRX1 is prevalent in inflammatory diseases leading to interest in NLRX1 as a drug target. There is a lack of structural information available for NLRX1 as only the leucine-rich repeat domain of NLRX1 has been crystallised. This lack of structural data limits progress in understanding function and potential druggability of NLRX1. We have modelled full-length NLRX1 by combining experimental, homology modelled and AlphaFold2 structures. The full-length model of NLRX1 was used to explore protein dynamics, mutational tolerance and potential functions. We identified a new RNA binding site in the previously uncharacterized N-terminus, which served as a basis to model protein-RNA complexes. The structure of the adenosine triphosphate (ATP) binding domain revealed a potential catalytic functionality for the protein as a member of the ATPase Associated with Diverse Cellular Activity family of proteins. Finally, we investigated the interactions of NLRX1 with small molecule activators in development, revealing a binding site that has not previously been discussed in literature. The model generated here will help to catalyse efforts towards creating new drug molecules to target NLRX1 and may be used to inform further studies on functionality of NLRX1.

Eukaryotic initiation factor 4B is a multi-functional RNA binding protein that regulates histone mRNAs.

RNA binding proteins drive proliferation and tumorigenesis by regulating the translation and stability of specific subsets of messenger RNAs (mRNAs). We have investigated the role of eukaryotic initiation factor 4B (eIF4B) in this process and identify 10-fold more RNA binding sites for eIF4B in tumour cells from patients with diffuse large B-cell lymphoma compared to control B cells and, using individual-nucleotide resolution UV cross-linking and immunoprecipitation, find that eIF4B binds the entire length of mRNA transcripts. eIF4B stimulates the helicase activity of eIF4A, thereby promoting the unwinding of RNA structure within the 5' untranslated regions of mRNAs. We have found that, in addition to its well-documented role in mRNA translation, eIF4B additionally interacts with proteins associated with RNA turnover, including UPF1 (up-frameshift protein 1), which plays a key role in histone mRNA degradation at the end of S phase. Consistent with these data, we locate an eIF4B binding site upstream of the stem-loop structure in histone mRNAs and show that decreased eIF4B expression alters histone mRNA turnover and delays cell cycle progression through S phase. Collectively, these data provide insight into how eIF4B promotes tumorigenesis.

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Microsatellite instability at U2AF-binding polypyrimidic tract sites perturbs alternative splicing during colorectal cancer initiation

BackgroundMicrosatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels.ResultsOur results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer.ConclusionsOverall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

PyRBDome: a comprehensive computational platform for enhancing RNA-binding proteome data.

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet, the extent of noise, including false positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome data in silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine-learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine-learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that although UV-cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.

TRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2.

The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a sub-complex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, these do not fully account for the importance of the Mtr4 Arch in TRAMP assembly. We further show that tRNA binding by single active-site subunits, Mtr4 and Trf4-Air2, differs from the double active-site TRAMP. TRAMP has reduced tRNA binding on the Mtr4 Fist and RecA2 domains, offset by increased tRNA binding on Air2 zinc knuckles 2 and 3. Competition between these RNA-binding sites may drive tRNA transfer between TRAMP subunits. We identify dynamic changes upon TRAMP assembly and RNA-recognition motifs that transfer RNA between TRAMP catalytic sites.

Multilevel gene expression changes in lineages containing adaptive copy number variants.

Copy-number variants (CNVs) are an important class of recurrent variants that mediate adaptive evolution. While CNVs can increase the relative fitness of the organism, they can also incur a cost. We previously evolved populations of Saccharomyces cerevisiae over hundreds of generations in glutamine-limited (Gln-) chemostats and observed the recurrent evolution of CNVs at the GAP1 locus. To understand the role that expression plays in adaptation, both in relation to the adaptation of the organism to the selective condition, and as a consequence of the CNV, we measured the transcriptome, translatome, and proteome of 4 strains of evolved yeast, each with a unique CNV, and their ancestor in Gln- conditions. We find CNV-amplified genes correlate with higher RNA abundance; however, this effect is reduced at the level of the proteome, consistent with post-transcriptional dosage compensation. By normalizing each level of expression by the abundance of the preceding step we were able to identify widespread divergence in the efficiency of each step in the gene in the efficiency of each step in gene expression. Genes with significantly different translational efficiency were enriched for potential regulatory mechanisms including either upstream open reading frames, RNA binding sites for SSD1, or both. Genes with lower protein expression efficiency were enriched for genes encoding proteins in protein complexes. Taken together, our study reveals widespread changes in gene expression at multiple regulatory levels in lineages containing adaptive CNVs highlighting the diverse ways in which adaptive evolution shapes gene expression.

Oligomerization regulates the interaction of Gemin5 with members of the SMN complex and the translation machinery

RNA-binding proteins are multifunctional molecules impacting on multiple steps of gene regulation. Gemin5 was initially identified as a member of the survival of motor neurons (SMN) complex. The protein is organized in structural and functional domains, including a WD40 repeats domain at the N-terminal region, a tetratricopeptide repeat (TPR) dimerization module at the central region, and a non-canonical RNA-binding site at the C-terminal end. The TPR module allows the recruitment of the endogenous Gemin5 protein in living cells and the assembly of a dimer in vitro. However, the biological relevance of Gemin5 oligomerization is not known. Here we interrogated the Gemin5 interactome focusing on oligomerization-dependent or independent regions. We show that the interactors associated with oligomerization-proficient domains were primarily annotated to ribosome, splicing, translation regulation, SMN complex, and RNA stability. The presence of distinct Gemin5 protein regions in polysomes highlighted differences in translation regulation based on their oligomerization capacity. Furthermore, the association with native ribosomes and negative regulation of translation was strictly dependent on both the WD40 repeats domain and the TPR dimerization moiety, while binding with the majority of the interacting proteins, including SMN, Gemin2, and Gemin4, was determined by the dimerization module. The loss of oligomerization did not perturb the predominant cytoplasmic localization of Gemin5, reinforcing the cytoplasmic functions of this essential protein. Our work highlights a distinctive role of the Gemin5 domains for its functions in the interaction with members of the SMN complex, ribosome association, and RBP interactome.

Splicing-specific transcriptome-wide association uncovers genetic mechanisms for schizophrenia

Recent studies have highlighted the essential role of RNA splicing, a key mechanism of alternative RNA processing, in establishing connections between genetic variations and disease. Genetic loci influencing RNA splicing variations show considerable influence on complex traits, possibly surpassing those affecting total gene expression. Dysregulated RNA splicing has emerged as a major potential contributor to neurological and psychiatric disorders, likely due to the exceptionally high prevalence of alternatively spliced genes in the human brain. Nevertheless, establishing direct associations between genetically altered splicing and complex traits has remained an enduring challenge. We introduce Spliced-Transcriptome-Wide Associations (SpliTWAS) to integrate alternative splicing information with genome-wide association studies to pinpoint genes linked to traits through exon splicing events. We applied SpliTWAS to two schizophrenia (SCZ) RNA-sequencing datasets, BrainGVEX and CommonMind, revealing 137 and 88 trait-associated exons (in 84 and 67 genes), respectively. Enriched biological functions in the associated gene sets converged on neuronal function and development, immune cell activation, and cellular transport, which are highly relevant to SCZ. SpliTWAS variants impacted RNA-binding protein binding sites, revealing potential disruption of RNA-protein interactions affecting splicing. We extended the probabilistic fine-mapping method FOCUS to the exon level, identifying 36 genes and 48 exons as putatively causal for SCZ. We highlight VPS45 and APOPT1, where splicing of specific exons was associated with disease risk, eluding detection by conventional gene expression analysis. Collectively, this study supports the substantial role of alternative splicing in shaping the genetic basis of SCZ, providing a valuable approach for future investigations in this area.

From Intercalation to External Binding: Ru(II) Complexes with a Spiro Ligand for TAR RNA Selective Binding and HIV-1 Reverse Transcriptase Inhibition.

As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.

Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

PUResNetV2.0: a deep learning model leveraging sparse representation for improved ligand binding site prediction

Accurate ligand binding site prediction (LBSP) within proteins is essential for drug discovery. We developed ProteinUNetResNetV2.0 (PUResNetV2.0), leveraging sparse representation of protein structures to improve LBSP accuracy. Our training dataset included protein complexes from 4729 protein families. Evaluations on benchmark datasets showed that PUResNetV2.0 achieved an 85.4% Distance Center Atom (DCA) success rate and a 74.7% F1 Score on the Holo801 dataset, outperforming existing methods. However, its performance in specific cases, such as RNA, DNA, peptide-like ligand, and ion binding site prediction, was limited due to constraints in our training data. Our findings underscore the potential of sparse representation in LBSP, especially for oligomeric structures, suggesting PUResNetV2.0 as a promising tool for computational drug discovery.

Transcriptome-wide mapping of small-molecule RNA-binding sites in live cells.

Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we describe the procedures for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells, identifying both the targets bound by the small molecule and the sites of binding therein. For complete details on the use and execution of this protocol, please refer to 1.

ASCC1 structures and bioinformatics reveal a novel helix-clasp-helix RNA-binding motif linked to a two-histidine phosphodiesterase

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE) (associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high resolution details and features conserved over 500 million years of evolution. Extending understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence–specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1 roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.