Evolution and Mechanistic Diversity of 5'-UTR G-quadruplex-Mediated Translation Regulation: Current Understanding and Methodological Challenges

RNA G-quadruplexes (rG4s) in 5'-UTRs represent complex regulatory elements capable of both inhibiting and activating mRNA translation through diverse mechanisms. This review analyzes the evolution of our understanding of 5'-UTR rG4-mediated translation regulation, from early discoveries of simple translation inhibitors to the current recognition of their multifaceted regulatory roles. We discuss canonical and non-canonical rG4 structures, their interactions with regulatory proteins including helicases and FMRP, and their function in both cap-dependent and IRES-mediated translation. Special attention is given to the synergistic effects between rG4s and upstream open reading frames (uORFs), stress-responsive translation regulation, and their role in repeat-associated non-AUG (RAN) translation linked to neurodegenerative diseases. We critically evaluate methodological challenges in the field, including limitations of current detection methods, reporter system artifacts, and the necessity to verify rG4 presence in endogenous transcripts. Recent technological advances, including genome editing and high-throughput sequencing approaches, have revealed that rG4 effects are more complex and context-dependent than initially thought. This review highlights the importance of developing more robust methodologies for studying rG4s at endogenous levels and carefully reevaluating previously identified targets, while emphasizing their potential as therapeutic targets in various diseases.