DATA_AVAILABILITY.docx

<i>ALKBH5 is a novel AGO2-binding protein involved in alternative splicing and miRNA binding</i>RNA binding proteins (RBPs) govern co- and post-transcriptional gene regulation by dynamically interacting with RNA targets. While RBPs are traditionally characterized by canonical RNA recognition motifs, more recently, RBPs lacking canonical motifs with diverse functions have been identified. ALKBH5, initially identified as an mRNA N6-methyladenosine (m6A) demethylase, is one such RBP with a yet-to-be-understood mechanism of RNA targeting.Our study explores the RNA binding properties of ALKBH5. By using combination of in vivo RNA Immunoprecipitation after Crosslinking (RIC), in vitro protein-RNA interaction assays, and HITS-CLIP analysis, we identify the C-terminus as crucial for RNA binding, shedding light on its target recognition mechanism. Furthermore, we unveil a novel interaction between ALKBH5 and AGO2, a prominent miRNA binding protein, suggesting a collaborative role in post-transcriptional regulation. Through knockdown experiments and integrated analyses of HITS-CLIP and RNAseq datasets, we uncover shared regulatory pathways and enriched splicing events between ALKBH5 and AGO2, emphasizing their potential synergy in RNA splicing. These findings not only deepen our understanding of ALKBH5's RNA binding properties but also unveil a novel dimension of its regulatory role in collaboration with AGO2, expanding the intricate network of post-transcriptional gene expression regulation.