Direct RNA sequencing and signal alignment reveal RNA structure ensembles in a eukaryotic cell

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Understanding RNA structure ensembles and their role in eukaryotic gene expression is crucial but remains incomplete.
• Existing methods have limitations in analyzing dynamic RNA structures at a single-molecule level.

Purpose of the Study:

• To develop and validate a novel method, sm-PORE-cupine, for identifying RNA structure ensembles.
• To investigate the relationship between RNA structure, gene expression, translation efficiency, and decay.

Main Methods:

• Coupling chemical probing with direct RNA sequencing for single-molecule RNA structure analysis (sm-PORE-cupine).
• Utilizing direct signal alignment and base mapping to enhance sequence mappability.
• Applying Bernoulli mixture model clustering for accurate separation of RNA structure ensembles.

Main Results:

• sm-PORE-cupine successfully identified isoform-specific structure ensembles in SARS-CoV-2 and the Candida albicans transcriptome.
• RNAs exhibit greater structural homogeneity in vitro, at elevated temperatures, and within 3' untranslated regions of C. albicans.
• Observed associations between RNA structure ensembles and altered translation efficiency and decay in C. albicans, validated by reporter assays.

Conclusions:

• sm-PORE-cupine is a powerful new tool for analyzing RNA structure and function across diverse transcriptomes.
• The study provides insights into how RNA structure ensembles dynamically regulate gene expression.
• Findings highlight the importance of RNA structural heterogeneity in biological processes.