Biology subjects

Velema, W.

Publications and source records attributed to Velema, W..

3 recordsLinked to original sources

bioRxiv2024

RNA secondary structure ensemble mapping in a living cell identifies conserved RNA regulatory switches and thermometers

RNA molecules can populate ensembles of alternative structural conformations, but comprehensively mapping RNA conformational landscapes within living cells presents significant challenges and has, as such, so far remained elusive. Here, we generated the first transcriptome-scale maps of RNA secondary structure ensembles in a living cell, using Escherichia coli as a model. Our analysis uncovered features of structurally-dynamic regions, as well as the existence of hundreds of highly-conserved bacterial RNA structural elements. Conditional structure mapping revealed extensive restructuring of RNA ensembles during cold shock, leading to the discovery of several novel RNA thermometers in the 5' UTRs of the cspG, cspI, cpxP and lpxP mRNAs. We mechanistically characterized how these thermometers switch structure in response to cold shock and revealed the cspE chaperone-mediated regulation of lpxP. Collectively, this work reveals a previously unappreciated complexity of RNA structural dynamics in living cells, and it provides a key resource to significantly accelerate the discovery of regulatory RNA switches.

molecular biology↗

bioRxiv2023

Siderophore-Mediated Conveyance of Antibacterial-Antisense Oligomers

Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Antibiotic antisense oligomers hold promise as powerful next-generation antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore-mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore-conjugated morpholino and PNA antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono-catechol group showed equally effective. Targeting the lacZ transcript resulted in dose-dependent decreased {beta}-galactosidase production, demonstrating selective protein downregulation. Whole-genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore-mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, our work provides a convenient approach for delivering antisense oligomers into bacteria.

microbiology↗

bioRxiv2021

Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a new method, Spatial 2-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a new strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

biochemistry↗