STORM-seq: a technology for simultaneous sequencing of twelve (and more) rRNA modifications

Miguel Angel Garcia-Campos

Schwartz Lab

Weizmann Institute of Science

RNA Biology, AWAJI, JAPAN�December, 2022

rRNA is richly modified

Reference

Yeast + Human

Taoka et al., 2018

RNA modifications in the rRNA can be highly dynamic!

• Archaeal hyperthermophile Thermococcus kodakarensis: whereas only 7 sites are modified at 55 °C, at its optimal growth temperature of 85 °C, up to 173 are acetylated.

Sas-Chen et al., 2020

Several RNA modification detection technologies�But we want to go High-Throughput and with Single-nucleotide resolution

• LC MS/MS
• 2D -Thin Layer Chromatography
• HPLC : High performance liquid chromatography
• Primer extensión
• Locus specific: RNAse-H, qPCR, electrospray ionization-mass spectrometry
• Next generation sequencing:
• Enzyme assisted
• Antibody-based
• Nanopore direct native RNA sequencing
• Chemical treatment
• Reverse transcription profiles

STORM-seq

• SimulTaneOus RNA Modifications sequencing (STORM-seq): is a method that integrates several RNAmod-seq methods into one methodology that is able to detect (currently) 12 different RNA modifications.
• 8 different library preparations
• 3 different RTases to generate
• 42 RNAmod metrics.

​

Joseph Georgeson

Example: Bisulfite sequencing

Example: CMC treatment

brainSTORM generate a profile for each nucleotide

• Up to 42 different metrics characterize each nucleotide.
• Making possible the discrimination from common and modified nucleotides.

Expected RNAmod signals

Unexpected RNAmod signals

m62A

m1acp3Y

New metrics detection: m62A

m62A

m62A

New metrics example:

m1acp3Y

m1acp3Y

Reference rRNA results

Thermococcus kodakarensis

• Thermophilic archaea.
• Isolated from a solftara/fumarole near the shore of Kodakarajima Island, Kagoshima, Japan.
• Optimal growth �temperature = 85 °C

Kodakara-Jima Island ©Wikicommons

Pyrococcus�furiosus

• Pyrococcus furiosus is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea.
• Optimum growth temperature = 100 °C

​

© Alfalo report by Michelle Kropf (Wikicommons)

Take home messages

• Chemical treatment and reverse transcription coupled to RNA-sequencing is able to generate accurate single-nucleotide RNA modification profiles.
• There are unexpected signals for RNA modification in already existing methods.
• It is clear that in archeal organisms RNA modifications status is sensitive to environmental cues.
• rRNA tuning and dynamics through RNA modifications might be the rule rather than the exception.

​

Resources

• Txtools: https://github.com/AngelCampos/txtools
• Schwartz lab GitHub Repo: https://github.com/SchwartzLab
• Website: https://angelcampos.github.io/

​

Thanks for your attention!

Funding