High-throughput functional profiling and evolutionary covariation analysis of entire riboswitch sequences

Area of Science:

• Molecular Biology
• Bioinformatics
• RNA Biology

Background:

• Riboswitches are RNA molecules known for structural dynamics crucial to cellular functions.
• Traditional sequence covariation analysis is limited to riboswitch aptamer domains, struggling with diverse expression platforms.

Purpose of the Study:

• To develop a novel bioinformatic approach for generating covariation models of complete riboswitch sequences.
• To expand the analysis of RNA structure dynamics beyond aptamer domains to include expression platforms.

Main Methods:

• Bioinformatically extending aptamer domains to include downstream sequences.
• Filtering sequences to identify bacterial intrinsic terminators using computational or experimental methods.
• Generating covariation models from filtered sequences, including high-throughput in vitro transcription and next-generation sequencing.

Main Results:

• Successfully generated a covariation model for the fluoride riboswitch consistent with its known mechanism.
• Developed covariation models for ZTP, lysine, and TPP riboswitches, supporting their previously established mechanisms.
• Demonstrated the method's efficacy in characterizing large numbers of riboswitch sequences.

Conclusions:

• The developed method enables comprehensive covariation modeling of entire riboswitches.
• This approach enhances the study of RNA structure dynamics and riboswitch evolution.
• Provides new insights into the mechanisms of various riboswitches.