Mechanism of Xrn1 duplex RNA unwinding

Abstract

RNA metabolism plays a central role in the regulation of gene expression, enabling cells to precisely control RNA abundance, quality, and function. In this context, RNA degradation is essential for maintaining cellular homeostasis, eliminating defective transcripts, and responding to environmental and developmental cues. The 5′–3′ exoribonuclease Xrn1 is a key enzyme in cytoplasmic RNA decay pathways and is known for its highly processive degradation activity. However, many physiological RNA substrates form stable secondary structures, raising a fundamental question of how Xrn1 efficiently processes structured RNA in the absence of a canonical helicase domain. In this study, I aimed to elucidate the molecular mechanism of RNA unwinding by Xrn1 using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) analysis. My results show that Xrn1 performs stepwise unwinding in units of approximately 8–9 base pairs (bp). Each unwinding event consists of multiple hidden sub-steps, during which mechanical strain is progressively accumulated prior to strand separation. These findings suggest that Xrn1 is not merely a degradative enzyme but possesses a mechanical mechanism that enables it to actively process structured RNA. I first investigated the function of conserved positively charged residues, Arg100 and Arg101, located near the 5′ phosphate-binding pocket. Mutations at these positions significantly reduced RNA degradation efficiency, with more pronounced effects observed for structured RNA substrates. These results indicate that Arg100 and Arg101 play critical roles in stabilizing RNA binding through electrostatic interactions and in maintaining directional translocation. Furthermore, smFRET analysis revealed that the stepwise unwinding pattern was preserved even in these mutants, suggesting that these residues primarily function to anchor the RNA substrate and facilitate strain accumulation rather than directly driving strand separation. Next, I examined the role of loop structures located along the RNA entry channel. Replacement of all loop regions with a flexible GGSGGS linker resulted in a substantial reduction in duplex RNA unwinding activity, while the ability to degrade single-stranded RNA was retained. This indicates that the loop structures are not required for catalytic activity but are essential for strand separation. Comparison of individual loop contributions revealed functional differences among them. Constructs containing Loop2 retained high unwinding activity, whereas those lacking Loop2 showed a significant reduction in activity. Constructs containing only Loop2 exhibited limited unwinding activity, while those containing only Loop1 or Loop3 showed little to no activity. These results indicate that Loop2 serves as the primary driver of strand separation, whereas Loop1 and Loop3 play supportive roles. Consistent with this, smFRET analysis showed that constructs containing only Loop2 exhibited unstable intermediate states, whereas those containing Loop1 and Loop3 displayed more stable and coordinated unwinding dynamics. This suggests that Loop2 promotes duplex destabilization, while Loop1 and Loop3 stabilize intermediate states and enhance unwinding efficiency. Further analysis of Loop2 revealed that its sequence, position, length, and conserved residues all contribute to its function. Substitution of the Loop2 sequence, relocation to alternative positions, or alteration of loop length all resulted in reduced unwinding activity. Mutations of conserved residues within Loop2 similarly impaired function. These results indicate that Loop2 is a finely tuned structural element that requires specific sequence features and precise spatial configuration for optimal activity. Finally, structural and sequence comparisons with Xrn2 revealed that Loop1 and Loop2 are evolutionarily conserved. This suggests that loop-mediated RNA processing is not unique to Xrn1 but may represent a conserved functional mechanism across the Xrn family. Taken together, my results indicate that Xrn1 achieves ATP-independent RNA duplex unwinding through the coordinated action of electrostatic interactions and structural elements. RK residues anchor the RNA substrate and enable strain accumulation, while Loop2 acts as a primary destabilizing element, supported by Loop1 and Loop3, which stabilize intermediate states and promote efficient unwinding. These findings provide a new mechanistic framework for understanding how exoribonucleases process structured RNA without helicase activity and expand our understanding of the physical principles underlying RNA degradation.