RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Investigating cis-regulatory sequences in RNA is crucial for understanding gene expression control.
• Existing methods may lack the sensitivity or dynamic range to comprehensively screen regulatory elements.
• The interplay between RNA sequences, codons, and translation efficiency requires robust screening tools.

Purpose of the Study:

• To develop and validate a novel, sensitive method (RNA-ID) for screening RNA cis-regulatory sequences.
• To demonstrate the method's capability in identifying known regulatory effects and discovering novel sequences.
• To establish a versatile platform for studying RNA-mediated and codon-mediated gene expression regulation.

Main Methods:

• Developed RNA-ID, a reporter system in yeast utilizing fluorescence-activated cell sorting (FACS).
• Employed a bidirectional GAL1,10 promoter driving expression of superfolder green fluorescent protein (GFP) and mCherry red fluorescent protein (RFP).
• Utilized ligation-independent cloning to construct large libraries for screening.

Main Results:

• RNA-ID demonstrated a dynamic range exceeding 250-fold with quantitative, dose-dependent responses.
• The method accurately recapitulated known translational inhibition by CGA codon pairs and stop codon read-through effects.
• High resolution enabled distinct population separation, and reproducible signals were observed across replicates.
• Evidence suggests the discovery of novel cis-regulatory sequences within short (9-nt) libraries.

Conclusions:

• RNA-ID is a robust and sensitive method for screening RNA cis-regulatory sequences.
• The platform is effective for studying both RNA-mediated and codon-mediated effects on gene expression.
• RNA-ID facilitates the discovery of novel regulatory elements in RNA, even within limited sequence spaces.