Development of technologies to portray the 3′Phospho-RNAome in translation and disease

Small RNAs are key regulators of gene expression and provide crucial insights into cellular states. RNA fragments, a subclass of small RNAs, are generated by nuclease cleavage of longer, structured RNA species (e.g., rRNA, long non-coding RNAs, snRNAs, and mRNAs). Depending on the nuclease involved, these fragments may carry a hydroxyl (3′-OH), phosphate (3′-P), or cyclic phosphate (2′-3′cP) group at their 3′ end. The latter two, known as 3′P RNAs, are highly conserved across life forms. While some fragments may result from RNA degradation, others act as bioactive molecules influencing cellular physiology. Specifically, 3′P-tRNA-derived fragments (3′P-tRFs) participate in cellular stress responses, regulate translation, and are associated with diseases like cancer and neurodegeneration. Their altered levels in disease suggest potential roles as biomarkers, therapeutic targets, or agents. Current limitations in sequencing and analytical methods challenge the comprehensive study of 3′P RNAs. This thesis addresses these challenges by developing novel technologies to characterize the 3′Phospho-RNAome, structured around three specific aims targeting distinct aspects of 3′P RNA biology. The first aim establishes an experimental approach for profiling 3′P RNA through DART-RNAseq (Direct Amplification of 3′ Phospho fragments RNA sequencing) and a qPCR assay (3′P-qPCR). The second aim investigates 3′P RNAs as molecular markers for Spinal Muscular Atrophy (SMA) and cutaneous squamous cell carcinoma (cSCC). SMA, a neurodegenerative disease caused by mutations in the SMN1 gene, has an urgent need for biomarkers to monitor disease progression and therapeutic responses. In a mouse model of severe SMA, DART-RNAseq identified 3′P RNAome profiles in healthy and diseased tissues, validating specific tRNA fragments. Further analyses of patient-derived fibroblasts before and after treatment identified 3′P RNAs associated with disease severity and treatment response. In cSCC, combining 3′P RNAome data from patient-derived cells and biopsies identified 3′P tRFs that distinguish healthy from tumour samples. Plasma validation highlighted these molecules’ potential as non-invasive biomarkers. The final aim evaluates 3′P tRNA fragments as ribosome-associated non-coding RNAs (rancRNAs) and molecular markers of cellular stress responses. Using DART-RNAseq and 3′P-qPCR in immortalized cell lines under oxidative stress, this chapter of the thesis identified 3′P-IleTAT as a ribosome-associated RNA marker of translational inhibition, which may actively suppress translation upon overexpression. Overall, this thesis provides a comprehensive technological framework for profiling 3′P RNAs, advancing our understanding of RNA-based regulation and its applications in clinical diagnostics and therapeutic development.