Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

TLDR

Circular RNAs in Drosophila, identified from over 100 RNA‑seq libraries and more than 10 billion reads, preferentially arise from long introns, are enriched for conserved miRNA sites in coding regions, accumulate mainly in neural tissues, and increase with age, underscoring their expanding role in the transcriptome. The study aims to rigorously annotate over 2,500 Drosophila circular RNAs using more than 5 billion paired‑end reads from >100 libraries across developmental stages, tissues, and cell lines. The authors performed comprehensive annotation of circular RNAs by analyzing >5 billion paired‑end reads from >100 diverse RNA‑seq libraries, enabling systematic identification of back‑splicing events. They found that most Drosophila circular RNAs arise from back‑splicing of protein‑coding genes, lack poly(A) tails, are evolutionarily conserved, harbor over 1,000 conserved miRNA seed sites mainly in coding exons, show neural‑gene bias and age‑dependent accumulation in CNS, making them a potential aging biomarker.

Abstract

Highlights•Fly circular RNAs were annotated from >100 libraries and >10 billion total RNA-seq reads•Circular RNA properties include strong preference for long flanking introns•Circular RNAs are strongly biased to contain coding region conserved miRNA sites•Circular RNAs dominantly accumulate in the nervous system and increase with ageSummaryCircularization was recently recognized to broadly expand transcriptome complexity. Circular RNAs are strongly biased to contain coding region conserved miRNA sites•Circular RNAs dominantly accumulate in the nervous system and increase with age Circular RNAs dominantly accumulate in the nervous system and increase with ageSummaryCircularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.Graphical abstract