Biology subjects

White, L. K.

Publications and source records attributed to White, L. K..

4 recordsLinked to original sources

bioRxiv2024

Nanopore sequencing of intact aminoacylated tRNAs

Transfer RNAs (tRNA) are decorated during biogenesis with a variety of modifications that modulate their stability, aminoacylation, and decoding potential during translation. The complex landscape of tRNA modification presents significant analysis challenges and to date no single approach enables the simultaneous measurement of important but disparate chemical properties of individual, mature tRNA molecules. We developed a new, integrated approach to analyze the sequence, modification, and aminoacylation state of tRNA molecules in a high throughput nanopore sequencing experiment, leveraging a chemical ligation that embeds the charged amino acid in an adapted tRNA molecule. During nanopore sequencing, the embedded amino acid generates unique distortions in ionic current and translocation speed, enabling application of machine learning approaches to classify charging status and amino acid identity. Specific applications of the method indicate it will be broadly useful for examining relationships and dependencies between tRNA sequence, modification, and aminoacylation.

molecular biology↗

bioRxiv2024

Quantification of subcellular RNA localization through direct detection of RNA oxidation

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, nucleus, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for the analysis of localized RNAs across different biological systems.

molecular biology↗

bioRxiv2024

Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

molecular biology↗

bioRxiv2022

Direct detection of RNA repair by nanopore sequencing

Ligation by plant and fungal RNA ligases yields an internal 2'-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA HAC1. The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2'-phosphorylated RNAs during nanopore sequencing. A 2'-phosphate at the splice junction of HAC1 mRNA inhibits 5'[->]3' degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2'-phosphorylated RNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events from multiple species.

molecular biology↗