Employing Tribe to Study RNA Interactions of Ataxin-2 in Drosophila S2 Cells

RNA-binding protein (RBP)-RNA interactions are fundamental for gene regulation and cellular homeostasis. Ataxin-2 is an RBP that has been shown to play an instrumental role in pathophysiological processes by binding to mRNA. Methods such as RNA immunoprecipitation (RIP), cross-linking immunoprecipitation (CLIP), and their variants can be used to study the interactions between Ataxin-2 and its targets, although their high sample requirements and labor-intensive workflows can limit their widespread use. RNA editing-based approaches, such as targets of RBPs identified by editing (TRIBE), provide effective alternatives. TRIBE enables transcriptome-wide identification of RBP targets by inducing site-specific adenosine-to-inosine (A-to-I) editing, which is subsequently detected through high-throughput RNA sequencing in both in vivo and in vitro systems. Compared to in vivo models, cell lines offer a rapid and flexible experimental design. Drosophila S2 cells are a commonly used insect cell line to investigate RNA-protein dynamics and serve as a versatile platform for studying RBP function. Here, we describe a protocol used for identifying RNA targets of Ataxin-2, a versatile RBP involved in post-transcriptional and translational regulation, in S2 cells using TRIBE. This method allows rapid, efficient, and reliable identification of Ataxin-2-associated RNA targets and can be readily applied to other RBPs. Key features • Streamlined workflow for identifying RNA targets of RBPs using TRIBE in Drosophila S2 cells. • TRIBE highlights the sites and nature of interactions between RBPs and RNA. • TRIBE overcomes the limitations of conventional RBP-RNA interaction studies like RIP, CLIP, and their variants.