Biology subjects

Yu, A. M.

Publications and source records attributed to Yu, A. M..

7 recordsLinked to original sources

bioRxiv2025

Massively parallel assay of human splice variants reveals cis-regulatory drivers of disease-associated and cell type-specific splicing regulation

Splice-disrupting variants (SDVs) underlie many human diseases, yet systematic functional maps of their effects across cell types remain limited. We developed Cell-type Oriented Massively Parallel reporter Assay of Splicing Signatures (COMPASS) to measure splicing outcomes for 87,546 single and double variants across more than 1,700 genes in five human cell lines of diverse tissue origin. COMPASS targets disease relevant gene sets, including ACMG actionable genes and SFARI autism-associated genes, enabling systematic dissection of splicing impacts in health and disease. Our measurements reveal numerous SDVs, including ClinVar variants currently classified as variants of uncertain significance. Using prime editing, we validate variant effects in the genome for ClinVar variants in BIN1, a gene implicated in Alzheimers disease, cancer, and cardiac pathology. Benchmarking our MPRA against state-of-the-art predictive models shows that while these approaches capture broad variant effects, important gaps remain. Our data reveal putative RNA-binding protein motifs whose disruption drives splicing changes, providing mechanistic insight into variant impact. Finally, cross-cell line comparisons reveal a distinct subset of variants that drive cell type-specific splicing programs. This study delivers the largest cell type-resolved, base-resolution atlas of splicing variant effects to date, providing a resource to support variant reclassification in clinical genomics and aid in the selection of therapeutic targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/681955v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@81765dorg.highwire.dtl.DTLVardef@181e55dorg.highwire.dtl.DTLVardef@f96eaeorg.highwire.dtl.DTLVardef@1d16d4a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 {degrees}C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 {degrees}C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

molecular biology↗

bioRxiv2023

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery

JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2- mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. As the Compound 2-resistant CS sequences, characterized by U/A-rich motifs, are prevalent in PASs from yeast to human, the CS region sequence may have more fundamental functions beyond determining drug resistance. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown and evolutionarily conserved sequence-specificity of the mRNA 3' processing machinery.

molecular biology↗

bioRxiv2021

Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data, and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamers P1 helix and the expression platforms transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

biochemistry↗

bioRxiv2018

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

MotivationRNA molecules can undergo complex structural dynamics, especially during transcription, which influence their biological functions. Recently developed high-throughput chemical probing experiments study RNA cotranscriptional folding to generate nucleotide-resolution reactivities for each length of a growing nascent RNA and reflect structural dynamics. However, the manual annotation and qualitative interpretation of reactivity across these large datasets can be nuanced, laborious, and difficult for new practitioners. We developed a quantitative and systematic approach to automatically detect RNA folding events from these datasets to reduce human bias/error, standardize event discovery, and generate hypotheses about RNA folding trajectories for further analysis and experimental validation.\n\nResultsDetection of Unknown Events with Tunable Thresholds (DUETT) identifies RNA structural transitions in cotranscriptional RNA chemical probing datasets. DUETT employs a feedback control-inspired method and a linear regression approach and relies on interpretable and independently tunable parameter thresholds to match qualitative user expectations with quantitatively identified folding events. We validate the approach by identifying known RNA structural transitions within the cotranscriptional folding pathways of the Escherichia coli signal recognition particle (SRP) RNA and the Bacillus cereus crcB fluoride riboswitch. We identify previously overlooked features of these datasets such as heightened reactivity patterns in the SRP RNA about 12 nucleotide lengths before base pair rearrangement. We then apply a sensitivity analysis to identify tradeoffs when choosing parameter thresholds. Finally, we show that DUETT is tunable across a wide range of contexts, enabling flexible application to study broad classes of RNA folding mechanisms.\n\nAvailabilityhttps://github.com/BagheriLab/DUETT\n\nContactjblucks@northwestern.edu, n-bagheri@northwestern.edu

bioinformatics↗

bioRxiv2018

Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

The series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways.\n\nHighlights- Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data\n- The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally\n- These structures rearrange dynamically to form an extended functional fold\n- Point mutations can disrupt and rescue cotranscriptional RNA folding pathways

biochemistry↗

bioRxiv2018

Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations.

Chemical probing experiments interrogate RNA structures by creating covalent adducts on RNA molecules in structure-dependent patterns. Adduct positions are then detected through conversion of the modified RNAs into complementary DNA (cDNA) by reverse transcription (RT) as either stops (RT-stops) or mutations (RT-mutations). Statistical analysis of the frequencies of RT-stops and RT-mutations can then be used to estimate a measure of chemical probing reactivity at each nucleotide of an RNA, which reveals properties of the underlying RNA structure. Inspired by recent work that showed that different reverse transcriptase enzymes show distinct biases for detecting adducts as either RT-stops or RT-mutations, here we use a statistical modeling framework to derive an equation for chemical probing reactivity using experimental signatures from both RT-stops and RT-mutations within a single experiment. The resulting formula intuitively matches the expected result from considering reactivity to be defined as the fraction of adduct observed at each position in an RNA at the end of a chemical probing experiment. We discuss assumptions and implementation of the model, as well as ways in which the model may be experimentally validated.

biochemistry↗