RNA Society Annual Conference 2024

The RNA modification N6-methyladenosine (m6A) is highly abundant in the human brain and implicated in neuropsychiatric and neurodegenerative disorders. However, most techniques for studying m6A cannot resolve modifications within RNA isoforms and we lack an isoform-level map of m6A sites in the brain. Profiling m6A within isoforms is therefore a critical step towards understanding the complex mechanisms that underpin brain function and disease. Oxford Nanopore direct RNA sequencing (DRS) can quantify isoform expression, modifications and polyA tail lengths, enabling simultaneous investigation of the transcriptome and epitranscriptome. We applied DRS to three post-mortem human brain regions: prefrontal cortex, caudate nucleus and cerebellum. We identified 57,000 m6A sites within 15,000 isoforms and estimated that >27% of mRNA molecules contained an m6A modification. Our results revealed both isoform- and brain- region-specific patterning of m6A modifications and polyA tail lengths. The prefrontal cortex exhibited a distinctive profile of specifically modified isoforms enriched in excitatory neuron cell types and also had the highest proportion of previously unannotated m6A sites. A population of isoforms were hypermodified with m6A and were associated with excitatory neuron cell types in all three brain regions. We also discovered >15k differentially expressed isoforms, >2k differentially modified m6A sites and 566 isoforms with differential polyA lengths between brain regions. Our study demonstrates the utility of DRS for investigating multiple features of RNA isoforms in the brain and provides new insights into brain region specificity and functioning with implications for neurological development and disease.

Lastly, researchers have been successfully DNase-treating flowcells to recover pores and increase yields from DNA runs for years. But can you wash and recover pores using the new RNA flowcells? I’ll present our preliminary results on the effect of washing RNA flowcells to increase DRS yields.

The lifecycle of an mRNA vaccine begins with its manufacture and formulation into lipid nanoparticles. The mRNA is then delivered to patients, where it is taken up by recipient cells and is translated into the encoded protein in the cytoplasm. This study uses Oxford Nanopore (ONT) sequencing to analyze each step in the mRNA vaccine lifecycle. We first show that ONT sequencing can be used during mRNA manufacturing to monitor mRNA quality and integrity, and to detect contaminating RNA species that can induce unwanted inflammatory responses. Direct RNA sequencing provides unbiased mRNA quality information as it detects the modified nucleotides used in mRNA vaccines, without Reverse Transcription or PCR amplification, which introduce biased errors. In addition, ONT sequencing can directly measure the impact of lipid nanoparticle formulation and storage on mRNA integrity. Finally, we describe the uptake, expression and final degradation of mRNA vaccines within cells. We sequence the mRNA vaccine within the recipient cell, and also investigate its broader impact on gene and protein expression. Together, this study uses ONT sequencing to trace the lifecycle of an mRNA vaccine, providing quality data from throughout the manufacturing process and insight into its mode of action within the cell.