Biology subjects

Irving, P. S.

Publications and source records attributed to Irving, P. S..

6 recordsLinked to original sources

bioRxiv2025

Ligand-binding pockets in RNA, and where to find them

RNAs are critical regulators of gene expression, and their functions are often mediated by complex secondary and tertiary structures. Structured regions in RNA can selectively interact with small molecules - via well-defined ligand binding pockets - to modulate the regulatory repertoire of an RNA. The broad potential to modulate biological function intentionally via RNA-ligand interactions remains unrealized, however, due to challenges in identifying compact RNA motifs with the ability to bind ligands with good physicochemical properties (often termed drug-like). Here, we devise fpocketR, a computational strategy that accurately detects pockets capable of binding drug-like ligands in RNA structures. Remarkably few, roughly 50, of such pockets have ever been visualized. We experimentally confirmed the ligandability of novel pockets detected with fpocketR using a fragment-based approach introduced here, Frag-MaP, that detects ligand-binding sites in cells. Analysis of pockets detected by fpocketR and validated by Frag-MaP reveals dozens of newly identified sites able to bind drug-like ligands, supports a model for RNA secondary structural motifs able to bind quality ligands, and creates a broad framework for understanding the RNA ligand-ome.

biophysics↗

bioRxiv2024

Structural determinants of inverted Alu-mediated backsplicing revealed by -MaP and -JuMP

Biogenesis of circular RNA usually involves a backsplicing reaction where the downstream donor site is ligated to the upstream acceptor site by the spliceosome. For this reaction to occur, it is hypothesized that these sites must be in proximity. Inverted repeat sequences, such as Alu elements, in the upstream and downstream introns are predicted to base-pair and represent one mechanism for inducing proximity. Here, we investigate the pre-mRNA structure of the human HIPK3 gene at exon 2, which forms a circular RNA via backsplicing. We leverage multiple chemical probing techniques, including the recently developed SHAPE- JuMP strategy, to characterize secondary and tertiary interactions in the pre- mRNA that govern backsplicing. Our data confirm that the antisense Alu elements, AluSz(-) and AluSq2(+) in the upstream and downstream introns, form a highly- paired interaction. Circularization requires formation of long-range Alu-mediated base pairs but does not require the full-length AluSq2(+). In addition to confirming long-range base pairs, our SHAPE-JuMP data identified multiple long-range interactions between non-pairing nucleotides. Genome-wide analysis of inverted repeats flanking circular RNAs confirm that their presence favors circularization, but the overall effect is modest. Together these results suggest that secondary structure considerations alone cannot fully explain backsplicing and additional interactions are key.

molecular biology↗

bioRxiv2023

Immune-induced remodelling of mRNA structurome regulates uORF-mediated translation

To survive stress, eukaryotes selectively translate stress-related transcripts while inhibiting growth-associated protein production. How this translational reprogramming occurs under biotic stress has not been systematically studied. To identify common features shared by transcripts with stress-upregulated translation efficiency (TE-up), we first performed high-resolution ribosome-sequencing in Arabidopsis during pattern-triggered immunity and found that TE-up transcripts are enriched with upstream open reading frames (uORFs). Under non-stress conditions, start codons of these uORFs (uAUGs) have higher-than-background ribosomal association. Upon immune induction, there is an overall downshift in ribosome occupancy at uAUGs, accompanied by enhanced translation of main ORFs (mORFs). Using in planta nucleotide-resolution mRNA structurome probing, we discovered that this stress-induced switch in translation is mediated by highly structured regions detected downstream of uAUGs in TE-up transcripts. Without stress, these structures are responsible for uORF-mediated inhibition of mORF translation by slowing progression of the translation preinitiation complex to initiate translation from uAUGs, instead of mAUGs. In response to immune induction, uORF-inhibition is alleviated by three Ded1p/DDX3X-homologous RNA helicases which unwind the RNA structures, allowing ribosomes to bypass the inhibitory uORFs and upregulate defence protein production. Conservation of the RNA helicases suggests that mRNA structurome remodelling is a general mechanism for stress-induced translation across kingdoms.

plant biology↗

bioRxiv2023

RNAvigate: Efficient exploration of RNA chemical probing datasets

Chemical probing technologies enable high-throughput examination of diverse structural features of RNA including local nucleotide flexibility, RNA secondary structure, protein- and ligand-binding, through-space interaction networks, and multi-state structural ensembles. Performing these experiments, by themselves, does not directly lead to biological insight. Instead, deep understanding of RNA structure-function relationships typically requires evaluating a system under structure- and function-altering conditions, linking these data with additional information, and visualizing multi-layered relationships. Current platforms lack the broad accessibility, flexibility, and efficiency needed to iterate on integrative analyses of these diverse, complex data. Here, we share the RNA visualization and graphical analysis toolset RNAvigate, a straightforward and flexible Python library. RNAvigate currently automatically parses twenty-one standard file formats (primary sequence annotations, per- and inter-nucleotide data, and secondary and tertiary structures) and outputs eighteen plot types. These features enable efficient exploration of nuanced relationships between chemical probing data, RNA structure, and motif annotations across multiple experimental samples. Compatibility with Jupyter Notebooks enables non-burdensome, reproducible, transparent and organized sharing of multi-step analyses and data visualization strategies. RNAvigate simplifies examination of multi-layered RNA structure information and accelerates discovery and characterization of RNA-centric functions in biology.

bioinformatics↗

bioRxiv2021

Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors

Many lncRNAs have been discovered using transcriptomic data, however, it is broadly unclear what fraction of lncRNAs are functional and what structural properties affect their phenotype. MUNC lncRNA, also known as DRReRNA, stimulates skeletal muscle differentiation. The prevailing hypothesis is that MUNC stimulates the Myod1 gene in cis as an enhancer RNA and stimulates expression of several other promyogenic genes in trans by recruiting the cohesin complex to their promoters. Experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by RNA structure prediction algorithms in the absence of experimental information. We discovered that these specific and structurally distinct domains are required for induction of different promyogenic genes, for binding at different genomic sites to regulate the expression of adjacent genes, and for binding the cohesin complex. Moreover, we found that induction of Myod1 or interaction with cohesin comprise only a subset of the broad regulatory impact of this lncRNA. Our study thus reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.

molecular biology↗

bioRxiv2019

RNA base pairing complexity in living cells visualized by correlated chemical probing

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

biochemistry↗