🧬 PubMed RNA Editing Daily Digest

RNA-based therapeutics offer versatile strategies for disease prevention and treatment, yet precise control over gene expression remains a major challenge. Self-amplifying RNA (saRNA), derived from the alphavirus genome, could be engineered to contain multiple subgenomic promoters, providing a unique RNA-only architecture for modular and independent regulation of downstream genes. However, the potential of saRNA as a programmable gene circuit enabling small-molecule-controlled on/off regulation of gene expression has remained largely unexplored. In this study, we engineered a series of saRNA constructs incorporating multiple regulatory modules, including a destabilizing domain (DD), the RNA-binding protein L7Ae, a tetracycline-responsive repressor (TetR), and kink-turn (k-turn) RNA motifs. This design allows individual downstream genes driven by distinct subgenomic promoters to be independently and reversibly regulated by the FDA-approved small-molecule ligands trimethoprim (TMP) or doxycycline (Dox). The engineered saRNAs were encapsulated into lipid nanoparticles, and saRNA-mediated expression of luciferase or fluorescent reporter proteins was systematically evaluated both in vitro and in vivo. Our results demonstrate that TMP and Dox function as effective molecular switches to autonomously turn on or off specific gene expression programs encoded within a single saRNA molecule. Collectively, this work establishes saRNA as a programmable RNA gene circuit platform with ligand-responsive, multi-gene regulatory capability, providing a versatile foundation for the development of controllable RNA therapeutics and synthetic biology applications.

STAMP (Surveying Targets by APOBEC-Mediated Profiling) employed the fusion protein between an RBP and the C-to-U editing enzyme APOBEC1 to mark and determine RBP's target transcripts. It exhibited wonderful performance in mammals, however, it was confirmed not working in flies and unclear its generality. In this study, APOBEC1 in STAMP was replaced with an APOBEC1-H122L/D124N mutant (APO1m), so that the method HyperSTAMP was established here in flies. HyperSTAMP method effectively and semi-quantitatively uncovered the targets of both Hrp48 (Heterogeneous nuclear ribonucleoprotein 48) and Thor in S2 cells with good signal to noise ratio, reproducibility, and consistency with other methods. It also captured RBP-binding positions on mRNAs through editing clusters on target RNAs. This study filled in the gap of lacking C-to-U editing enzyme in insects and provided a good tool for the experiments of double labeling RNA when exploring multiple RBPs concurrently.

Adenosine-to-inosine (A-to-I) mRNA editing can alter protein sequence and function, enabling bacteria to express two RNA and protein versions encoded by the same gene. However, its prevalence and significance in clinical bacterial settings remain unclear. We collected ten Escherichia coli and seven Pseudomonas aeruginosa isolates from hospitalized patients with urinary tract infections (UTI) or ear infections. Whole-genome and transcriptome sequencing were performed for each isolate, followed by Sanger sequencing for selected sites. We present the first comprehensive analysis of A-to-I RNA editing in pathogenic bacteria isolated from hospitalized patients. We identified dozens of A-to-I RNA editing sites, including novel sites not previously reported in nonpathogenic E. coli and P. aeruginosa strains. We found that E. coli exhibits higher editing levels and a greater number of editing sites than P. aeruginosa. Most editing sites are embedded within a conserved 7-base motif and are frequently located in predicted stem-loop RNA secondary structures, highlighting the importance of both sequence and structure for editing site recognition in both the examined species. Most editing events occur in mRNA and often result in nonsynonymous amino acid changes, with a notable prevalence of tyrosine-to-cysteine substitutions. Finally, we observed that editing patterns are similar between antibiotic-resistant and sensitive isolates, suggesting a more general role in the biology of the examined species. Adenosine-to-inosine RNA editing is a feature of pathogenic bacteria isolated from clinical samples. Our findings expand current knowledge of bacterial RNA editing in clinical contexts and provide a framework for future functional investigations.