Grad-seq guides the discovery of ProQ as a major small RNA-binding protein.

Area of Science:

• * Molecular Biology
• * Genomics
• * Microbiology

Background:

• * Next-generation RNA sequencing aids transcriptome annotation but struggles with noncoding RNA (ncRNA) function prediction due to sequence and structural diversity.
• * Functional RNA identification requires criteria beyond primary sequence, such as RNA-protein interactions.
• * Understanding the RNA landscape is crucial for microbiome and environmental studies.

Purpose of the Study:

• * To develop and apply a method for functional RNA landscape analysis based on RNA-protein interactions.
• * To identify novel ncRNA classes and RNA-binding proteins in *Salmonella enterica*.
• * To characterize the role of the protein ProQ in RNA regulation.

Main Methods:

• * Gradient profiling by sequencing (Grad-seq) was used to analyze the full ensemble of cellular RNAs in *Salmonella enterica*.
• * Transcripts were partitioned based on their RNA-protein interaction networks.
• * RNA-binding protein ProQ and its associated RNAs were identified and characterized.

Main Results:

• * Grad-seq successfully captured established RNA classes and enabled the discovery of a large collection of structured small RNAs.
• * These small RNAs form stable complexes with the conserved protein ProQ.
• * ProQ was identified as an abundant RNA-binding protein influencing *Salmonella* gene expression globally.

Conclusions:

• * Grad-seq provides a powerful, sequence-independent approach to define functional RNA classes and RNA-binding proteins.
• * The discovery of ProQ-bound small RNAs expands our understanding of bacterial ncRNA biology.
• * This method is applicable to both model organisms and genetically challenging species.