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Surovtseva, Y. V.

Publications and source records attributed to Surovtseva, Y. V..

9 recordsLinked to original sources

Distillation enables scalable high-fidelity virtual screening across ultra-large chemical libraries

Accurate virtual screening of ultra-large chemical libraries remains challenging. Existing approaches rely on lower-fidelity scoring functions or sampling-based strategies that can limit predictive accuracy and bias the exploration of chemical space. Here, we present FastBindRank, a distillation-based framework that transfers the predictive power of the structure-based model Boltz-2 into an efficient sequence-based surrogate. Trained on [~]1% of the 122-million-compound PubChem library, FastBindRank enables high-fidelity screening at scale. Applied to histone deacetylase 11 (HDAC11), FastBindRank substantially enriched high-confidence binders relative to the background chemical space. The lightweight model captured structural patterns associated with predicted binding, revealing structural determinants of binding. Under a comparable computational budget, FastBindRank achieved a 74-fold increase in hit rate and over a 30-fold increase in discovery yield over direct subset-based screening. Experimental validation confirmed the activity of two novel compounds. These results establish distillation as a practical strategy for scalable, high-fidelity virtual screening of ultra-large chemical libraries.

bioinformatics↗

Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders

Aberrant biomolecular condensates are implicated in neurological disorders including ALS, frontotemporal dementia, and DYT1 dystonia, yet approaches to systematically identify their modulators remain limited. Here we establish MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatic pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1,760 FDA-approved compounds in a cellular DYT1 dystonia model, we identify drugs that alter aberrant condensate properties, validating the platform for condensate-targeted drug discovery. In parallel, a genome-wide CRISPR/Cas9 screen links condensate accumulation to microcephaly genes and more than 10 additional neurodevelopmental disorders. Machine learning and confocal imaging resolves distinct condensate phenotypes: loss of microcephaly-associated ZNF335 results in nucleoplasmic condensates, whereas RNF26 deletion produces nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable resource for identifying corrective modulators of condensates and establishes a link between nuclear condensate accumulation and neurodevelopmental disorders.

cell biology↗

The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

Cancer initiates as a consequence of genomic mutations, and its subsequent progression relies on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. To form more established connections between these two cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratorys established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. We show that APOBEC3A is required for cell cycle progression and global protein synthesis. More specifically, we highlight APOBEC3As role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. Through an innovative nuclear RNA sequencing methodology, we identify candidate APOBEC3A C-to-U editing sites on the pre-rRNA and pre-mRNAs for the first time. Our work reveals the exciting possibility that the pre-rRNA can be edited during its maturation. More broadly, we found an additional function of APOBEC3A in cancer pathology, expanding its relevance as a target for cancer therapeutics.

molecular biology↗

Discovery of novel microRNA regulators of ribosome biogenesis

While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2,603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits included 27 well-conserved microRNAs present in MirGeneDB, and were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, while causing diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We discovered that the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, directly and potently target the ribosomal protein mRNA RPS28 via tandem primate-specific 3 UTR binding sites, causing a severe pre-18S pre-rRNA processing defect. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/526327v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1449771org.highwire.dtl.DTLVardef@329602org.highwire.dtl.DTLVardef@1168c97org.highwire.dtl.DTLVardef@1684da9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Human Nucleolar Protein 7 (NOL7) is required for pre-rRNA transcription and pre-18S rRNA processing

The main components of the essential cellular process of eukaryotic ribosome biogenesis are highly conserved from yeast to humans. Among these, the transcription-U3 Associated Proteins (t-UTPs) are a small subunit processome subcomplex that coordinate the first two steps of ribosome biogenesis in transcription and pre-18S processing. While we have identified the human counterparts of most of the yeast Utps, the homologs of yeast Utp9 and Bud21 (Utp16) have remained elusive. In this study, we find NOL7 is the likely ortholog of Bud21. Previously described as a tumor suppressor through regulation of antiangiogenic transcripts, we now show that NOL7 is required for early pre-rRNA stability and pre-18S processing in human cells. These roles lead to decreased protein synthesis, induction of the nucleolar stress response, and defects in cell cycle progression upon NOL7 depletion. Beyond Bud21s nonessential role in yeast, we establish human NOL7 as an essential UTP that is necessary for both pre-rRNA transcription and processing.

molecular biology↗

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for ribosome biogenesis, and as an interactor with the PeBoW complex (PES1, BOP1, WDR12) in high-throughput affinity purifications. The PeBoW complex has been shown to be required for pre-28S rRNA processing. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in mammalian cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We also demonstrate that RSL24D1 is physically complexed with RNA polymerase I, revealing a connection between large ribosomal subunit biogenesis and rDNA transcription. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large subunit biogenesis factors in pre-rRNA transcription control.

biochemistry↗

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis, and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting ribosome biogenesis. However, there is a need for a more direct high-throughput assay for nucleolar function to further evaluate hits. Previous reports have monitored nucleolar RNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay for nucleolar function which enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay utilizes two siRNA controls, a negative non-targeting siRNA control (siNT) and a positive siRNA control targeting POLR1A (siPOLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dissociation. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of ribosome biogenesis in health and disease.

cell biology↗

Restriction of SARS-CoV-2 Replication by Targeting Programmed -1 Ribosomal Frameshifting In Vitro

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed -1 ribosomal frameshifting (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting -1 PRF as an effective antiviral strategy for SARS-CoV-2.

molecular biology↗

Model colibactins exhibit human cell genotoxicity in the absence of host bacteria

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wildtype pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action - DNA alkylation versus crosslinking - in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the observed bacteria-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an E. coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with moduleskipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand crosslinking in the native pathway.

cancer biology↗