Biology subjects

Keane, S. C.

Publications and source records attributed to Keane, S. C..

11 recordsLinked to original sources

bioRxiv2026

Localized ribosome access and distal tuning via the Listeria prfA RNA thermometer

RNA thermometers (RNATs) are temperature-responsive structures in 5' untranslated regions (UTRs) of bacterial messenger RNA (mRNA) that control translation by modulating ribosome access. The Listeria monocytogenes prfA RNAT represses translation of PrfA (positive regulatory factor A), the master virulence regulator, at ambient temperature and activates it near the human host temperature ([~]37 {degrees}C) by modulating ribosome binding site (RBS) accessibility. However, the prfA RNAT shares no homology with known RNAT classes, and its unfolding mechanism remains unclear. Here, we used analytical ultracentrifugation and single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) to map prfA RNAT unfolding. SiM-KARTS analysis demonstrates that thermal opening occurs predominantly at the RBS, while the upper helix of the RNAT hairpin remains largely folded at 37 {degrees}C. The RBS binding kinetics increases with temperature in parallel with translation output, establishing a quantitative link between structural unfolding and function. Mutations in the upper helix impair thermal regulation, indicating that this region tunes switching even as it stays structured at host temperature. Together, these data reveal a hierarchical unfolding pathway in which initial RBS opening triggers activation, whereas the upper helix remotely tunes temperature sensitivity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/717274v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a95daborg.highwire.dtl.DTLVardef@144bc42org.highwire.dtl.DTLVardef@1a39bf6org.highwire.dtl.DTLVardef@545e60_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Integrated in silico and experimental screening identifies novel ligands that target precursor microRNA-31 at the Dicer cleavage site

MicroRNAs (miRNAs) regulate gene expression and the dysregulation in mature miRNA levels has been implicated in a wide variety of diseases. In particular, altered levels of mature microRNA-31 (miR-31) has been linked to a variety of different cancers. Targeting functionally relevant sites of the precursor structure of miR-31 with small molecules offer a strategy to regulate miR-31 maturation. Herein we describe a virtual screening approach to explore the druggability of the precursor structure of microRNA-31 (pre-miR-31). We used a structure-guided approach to virtually screen a fragment library and followed up with experimental characterization of top-ranking candidates, leading to the identification of several compounds that bound to pre-miR-31. Further characterization of the RNA-ligand complexes by heteronuclear single quantum coherence (HSQC) NMR spectroscopy revealed three compounds bound pre-miR-31 at the Dicer cleavage site, suggesting that these compounds may function to inhibit Dicer processing. Using these initial hits, we performed chemical structure similarity searches and identified additional binders of pre-miR-31 that had equivalent or enhanced binding relative to the parent compounds. These studies suggest a generalizable approach by which RNA-binding ligands can be identified from large chemical databases. These hits can then be further optimized to improve affinity and specificity for downstream functional assays.

biophysics↗

bioRxiv2025

Structural features within precursor microRNA-20a regulate Dicer-TRBP processing

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression of target messenger (m) RNAs. To maintain proper miRNA expression levels, the enzymatic processing of primary and precursor miRNA elements must be strictly controlled. However, the molecular determinants underlying this strict regulation of miRNA biogenesis are not fully understood. Here, we determined the solution structure of pre-miR-20a, an oncogenic miRNA and component of the oncomiR-1 cluster, using nuclear magnetic spectroscopy (NMR) spectroscopy and small angle X-ray scattering (SAXS). Our structural studies informed on key secondary structure elements of pre-miR-20a which may control its enzymatic processing, namely a flexible apical loop and single-nucleotide bulge near the dicing site. We found that alternative conformations within pre-miR-20as apical loop function to self-regulate its Dicer-TRBP processing, and that a single nucleotide bulge at the -5 position from the 5'-cleavage site is critical for efficient processing. We additionally found that a disease-related single-nucleotide polymorphism in pre-miR-20a, predicted to disrupt the structure near the dicing site, resulted in reduced processing. These results further our structural understanding of the oncomiR-1 cluster and show how transient RNA conformers can function to self-regulate maturation.

biochemistry↗

bioRxiv2025

In silico discovery of compounds targeting NPSL2: a regulatory element in the human oncomiR-1 primary microRNA.

NPSL2 is a stem loop element within the oncomiR-1 polycistronic primary microRNA (miRNA) cluster. NPSL2 is predicted to mediate conformational rearrangements within oncomiR-1 to regulate the biogenesis of certain miRNA elements within the cluster. The regulatory role of NPSL2 makes it a promising target for small molecule ligands, which we explored through structure-based small molecule ligand discovery. Starting with an NMR-derived ensemble of NPSL2 structures, we identified ligandable cavities within NPSL2 using RNACavityMiner. We then used molecular docking to screen the ZINC library against these cavities to identify potential small molecule hits. A sampling of commercially available compounds from the initial hits were purchased for experimental characterization by saturation transfer difference (STD) and heteronuclear single quantum coherence (HSQC) NMR spectroscopy. This approach led to the identification and characterization of at least eight compounds that bind preferentially within the internal loop of NPSL2. Importantly, this workflow, which combined virtual screening and experimental validation, can be used to identify small molecules that bind to any known RNA three-dimensional structure.

biophysics↗

bioRxiv2024

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

biophysics↗

bioRxiv2024

Functional Validation of SAM Riboswitch Element A from Listeria monocytogenes

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SAM is essential to all domains of life, serving as a ubiquitous methyl donor and mediator of trans-sulfuration. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine, a sulfur containing amino acid and substrate for sulfur metabolism. SreA is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a similar structure and SAM binding properties to other well characterized SAM-I riboswitches. Interestingly, SreA regulates transcription at a distinctly lower (nM) ligand concentration than other SAM riboswitches but does not substantially terminate transcription, even in the presence of mM SAM. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/596223v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@16b3af4org.highwire.dtl.DTLVardef@eb6aecorg.highwire.dtl.DTLVardef@1b8e7ecorg.highwire.dtl.DTLVardef@823cf7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Use of steric blocking antisense oligonucleotides for the targeted inhibition of junction containing precursor microRNAs

Antisense oligonucleotides (ASOs) are widely used as therapeutics for neurodegenerative diseases, cancers, and virus infections. One class of ASOs functions to enhance protein expression by sequestering the mature microRNA (miRNA) in a double-stranded structure within the RNA-induced silencing complex (RISC). An alternative approach for the targeted control of gene expression is to use ASOs that bind to the pre-elements of miRNAs (pre-miRNAs) and modulate their enzymatic processing. Here, we demonstrate that ASOs can be used to disrupt a specific structural feature, "junction," within pre-miR-31 that is important in directing efficient processing by the Dicer/TRBP complex. Furthermore, we extend and validate this strategy to pre-miR-144, which has a similar junction-dependent structure-function relationship. We found that a significant number of human pre-miRNAs are predicted to contain junctions, and validated our ASO approach on several members of this group. Importantly, we also verified the application of junction-targeting ASOs for the specific inhibition of pre-miRNA processing in cell. Our study reemphasizes the important roles of RNA structure in regulating Dicer/TRBP processing of pre-miRNAs and provides the framework to develop structure-informed ASOs that serve to inhibit miRNA production.

biochemistry↗

bioRxiv2023

Structure of pre-miR-31 reveals an active role in Dicer processing

As an essential post-transcriptional regulator of gene expression, microRNA (miR) levels must be strictly maintained. The biogenesis of many, but not all, miRs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers and interestingly, its biogenesis is not known to be regulated by protein binding partners. Therefore, the intrinsic structural properties of pre-miR-31 can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of the precursor element of miR-31 (pre-miR-31) to investigate the role of distinct structural elements in regulating Dicer processing. We found that the presence or absence of mismatches within the helical stem do not strongly influence Dicer processing of the pre-miR. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by Dicer. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influence both Dicer binding and processing. Our results enrich our understanding of the active role that RNA structure plays in regulating Dicer processing which has direct implications for control of gene expression.

biophysics↗

bioRxiv2022

Solution structure of NPSL2, a regulatory element in the oncomiR-1 RNA

The miR-17~92a polycistron, also known as oncomiR-1, is commonly overexpressed in multiple cancers and has several oncogenic properties. OncomiR-1 encodes six constituent microRNAs (miRs), each enzymatically processed with different efficiencies. However, the structural mechanism that regulates this differential processing remains unclear. Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a structure that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing. Here, we determined the solution structure of NPSL2 using solution NMR spectroscopy. This is the first high-solution structure of an oncomiR-1 element. NPSL2 adopts a hairpin structure with a large, but highly structured, apical and internal loops. The 10-bp apical loop contains a pH-sensitive A+{middle dot}C mismatch. Additionally, several adenosines within the apical and internal loops have elevated pKa values. The protonation of these adenosines can stabilize the NPSL2 structure through electrostatic interactions. Our study provides fundamental insights into the secondary and tertiary structure of an important RNA hairpin proposed to regulate miR biogenesis.

biophysics↗

bioRxiv2021

pH dependence of C·A, G·A and A·A mismatches in the stem of precursor microRNA-31

MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A*A, G*A, and C*A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/465784v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@fec8a8org.highwire.dtl.DTLVardef@1279235org.highwire.dtl.DTLVardef@efdd58org.highwire.dtl.DTLVardef@1acf54e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

NMR chemical shift assignments of RNA oligonucleotides to expand the RNA chemical shift database

RNAs play myriad functional and regulatory roles in the cell. Despite their significance, three-dimensional structure elucidation of RNA molecules lags significantly behind that of proteins. NMR-based studies are often rate-limited by the assignment of chemical shifts. Automation of the chemical shift assignment process can greatly facilitate structural studies, however, accurate chemical shift predictions rely on a robust and complete chemical shift database for training. We searched the Biological Magnetic Resonance Data Bank (BMRB) to identify sequences that had no (or limited) chemical shift information. Here, we report the chemical shift assignments for 12 RNA hairpins designed specifically to help populate the BMRB.

biophysics↗