Biology subjects

Jones, A. N.

Publications and source records attributed to Jones, A. N..

7 recordsLinked to original sources

bioRxiv2026

Structural and biophysical insights into the unique RNase YicC

RNA cleavage and processing are highly conserved regulatory mechanisms across bacteria. We recently discovered a novel family of RNases called YicC, conserved across all bacteria but unrelated to known RNase families. The cryo-EM structure of RNA bound to Escherichia coli YicC endonuclease suggested a clamshell-like ribonuclease mechanism for cleavage, although its exact mechanism and function remain elusive. Here, we report new cryo-EM structures that provide snapshots of the closing of the complex and also capture a surprising dimeric-RNA:protein complex. We further characterize the RNA cleavage targets in solution using nuclear magnetic resonance (NMR) spectroscopy, and use all-atom molecular dynamics (MD) simulations to study Mg2+ binding. Our findings suggest that the YicC family can bind RNA hairpin structures and dimeric conformations with appropriate sequence and structural constraints, with cleavage driven strongly by Mg2+ ion localization. This study therefore provides overall insight into the novel cleavage mechanism of the highly conserved family of YicC-like endoribonucleases.

biochemistry↗

bioRxiv2024

RNA Chemical Probing Reagents and Protein Amino Acids: a Double-Edged Sword

RNA chemical probing experiments are a broadly used method for revealing the structure of RNA, as well as for identifying protein binding sites. This is beneficial for expanding our understanding of biological processes governed by protein-RNA complex interactions, as well as facilitating the identification of complex inhibiting molecules. The reagents commonly used in chemical probing experiments are highly reactive, methylating or acylating flexible RNA nucleotides. The highly reactive nature of the chemical probes means that they can also react with nucleophilic amino acid side chains, and subsequently affect protein-RNA binding events. We combine molecular dynamics (MD) simulations, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and nuclear magnetic resonance (NMR) experiments to show that commonly used RNA chemical probes react with protein amino acids, and demonstrate that this effect alters protein-RNA binding interactions through binding shift assays. We discuss the implications of this phenomenon in elucidating the protein-RNA interaction interface using chemical probing experiments. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/586119v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@13378baorg.highwire.dtl.DTLVardef@3b0114org.highwire.dtl.DTLVardef@1c3f967org.highwire.dtl.DTLVardef@12afaec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Cooperativity in RNA chemical probing experiments modulates RNA 2D structure

Small molecule chemical probes that covalently bond atoms of flexible nucleotides are widely employed in RNA structure determination. Atomistic molecular dynamic (MD) simulations recently revealed that the binding of RNA by chemical probes is influenced by cooperative effects, leading to measured reactivities that depend on the concentration of the chemical probe. In this work, we used selective 2 hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing experiments to explore whether RNA structures are modulated by chemical probe binding events. We find that as the concentration of a chemical probe increases, modified nucleotides locally modulate the RNA structure, resulting in the increase or decrease of chemical probe reactivity in surrounding nucleotides. This cooperative effect is dependent on both chemical probe concentration and size. We find that these cooperative effects can be used to link structurally related nucleotides, and that the cooperative effects result in strikingly different 2D structure predictions as probe concentrations are varied.

biochemistry↗

bioRxiv2022

Cotranscriptional folding of the lncRNA Xist A-repeats indicates a modular structure

LncRNAs are emerging to play crucial roles in the regulation of many essential cellular processes and have been linked to human disease, but a detailed understanding of their structure and how this relates to underlying molecular mechanisms is still limited. The structure that a lncRNA adopts can interconvert between multiple conformations. However, characterizing the structure and dynamics is challenging given their large size. Here, we present an integrated approach, combining biochemical and biophysical techniques to investigate the core structural elements and conformational dynamics of the A-repeats of the lncRNA Xist. We combine chemical RNA structure probing, SAXS, NMR-spectroscopy and cryo-EM to comprehensively describe the conformational landscape of the Xist A-repeats. We show that under native-like conditions, the A-repeats are modular, comprising building blocks made from stable AUCG tetraloop hairpins and inter-repeat dimers separated by flexible uracil-rich regions. The structural core of the A-repeats involves dimerization of sequential repeats to form two subdomains, comprising repeats 1-4 and 5-8. The overall topology of the A-repeats is dynamic, with structural variability linked to the uracil-rich linker regions. Our results rationalize context and buffer-dependent structural variations of the Xist lncRNA. The integrative approach presented here establishes a general pipeline for investigating lncRNA structure and dynamics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/501616v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1761cbeorg.highwire.dtl.DTLVardef@15905e0org.highwire.dtl.DTLVardef@190bf0corg.highwire.dtl.DTLVardef@1eeca5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

microbiology↗

bioRxiv2021

Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 M, 8.5 M, 24.1 M, 8.2 M and 13.6 M, respectively. A separate experiment focusing on a combination of 10 M stenoparib and 0.5 M remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

microbiology↗

bioRxiv2021

Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

Alternative splicing is controlled by differential binding of trans-acting RNA binding proteins (RBPs) to cis-regulatory elements in intronic and exonic pre-mRNA regions 1-3. How secondary structure in the pre-mRNA transcripts affects recognition by RBPs and determines alternative exon usage is poorly understood. The MALT1 paracaspase is a key component of signaling pathways that mediate innate and adaptive immune responses 4. Alternative splicing of MALT1 exon7 is critical for controlling optimal T cell activation 5,6. Here, we demonstrate that processing of the MALT1 pre-mRNA depends on RNA structural elements that shield the 5 and 3 splice sites of the alternatively spliced exon7. By combining biochemical analyses with chemical probing and NMR we show that the RBPs hnRNP U and hnRNP L bind competitively and with comparable affinities to identical stem-loop RNA structures flanking the 5 and 3 splice sites of MALT1 exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L unwinds these RNA elements to facilitate recruitment of the essential splicing factor U2AF2 to promote exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.

biochemistry↗