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

Area of Science:

• Molecular Biology
• RNA Biology
• Bioinformatics

Background:

• Riboswitches are RNA molecules that regulate gene expression through structural rearrangements.
• Sequence covariation analysis is effective for studying riboswitch aptamer domains but challenging for diverse expression platforms.
• Understanding the full riboswitch structure is crucial for elucidating cellular functions and RNA dynamics.

Purpose of the Study:

• To develop a novel approach for identifying complete transcriptional riboswitch sequences, including expression platforms.
• To generate covariation models for entire riboswitches by integrating aptamer and expression platform sequences.
• To expand the understanding of riboswitch mechanisms and the evolution of RNA structure dynamics.

Main Methods:

• Bioinformatically extending conserved aptamer domains to capture potential expression platforms.
• Filtering candidate sequences using computational prediction of intrinsic terminators or a high-throughput functional assay.
• Developing full riboswitch sequence covariation models using filtered sequences.

Main Results:

• Successfully characterized 1901 fluoride riboswitch sequences using the high-throughput assay.
• The prediction filtering approach effectively identified novel, highly functional fluoride riboswitch variants with few false positives.
• Covariation models for ZTP, lysine, and TPP riboswitches supported previously proposed rearrangement mechanisms.

Conclusions:

• The developed hybrid computational and high-throughput experimental method enables characterization of large numbers of riboswitch sequences.
• This approach facilitates the generation of new covariation models for complete riboswitches.
• The findings enhance the understanding of riboswitch mechanisms and RNA structure dynamics evolution.