Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding

Area of Science:

• Molecular Biology
• Synthetic Biology
• Post-transcriptional Regulation

Background:

• Gene-regulatory networks require both inhibitory and upregulatory elements.
• Most known RNA-based regulatory elements are inhibitory, limiting the design of complex networks.
• Understanding RNA-binding protein (RBP)-RNA interactions is crucial for post-transcriptional control.

Purpose of the Study:

• To explore the regulatory effects of RBP-RNA interactions in bacterial 5' UTRs using synthetic biology.
• To identify both inhibitory and upregulatory RNA-based regulatory modules.
• To provide a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

Main Methods:

• Utilized a synthetic biology approach combined with SHAPE-seq (Selective 2′-hydroxyl acylation analyzed by primer extension sequencing).
• Constructed a library of RNA hairpins positioned upstream of a reporter gene.
• Co-expressed RNA hairpins with their matching RBPs to observe regulatory responses.

Main Results:

• Observed diverse regulatory responses, including translational stimulation, repression, and cooperative behavior.
• Identified three distinct in vivo states of RNA molecules: molten (translation-ready), structured (translation-inhibited), and semi-structured (partially functional in the presence of RBPs).
• Demonstrated that RBPs can modulate RNA structure to control translation.

Conclusions:

• RBP-RNA interactions play a significant role in post-transcriptional gene regulation.
• The study provides novel insights into the mechanisms of RNA-based gene control.
• The developed approach serves as a foundation for engineering synthetic gene-regulatory circuits.