UPF1, a conserved RNA helicase and core effector of nonsense-mediated mRNA decay (NMD), plays key roles in post-transcriptional gene regulation. UPF1 loss is lethal in most eukaryotes, restricting in-depth study of its non-canonical molecular and biological functions. In Arabidopsis thaliana, we demonstrate that this lethality primarily arises from autoimmunity. By suppressing immune signalling in eds1 or pad4 backgrounds, we generated viable upf1 null mutants. The upf1 eds1 plants exhibit developmental phenotypes such as slow growth, delayed flowering, reduced vigour, and partial sterility. However, they consistently display better overall growth than upf1 pad4 plants. This observation aligns with our transcriptome data, which indicates stronger suppression of immune response in the eds1 background. Importantly, the reduced immune activation in upf1 eds1 plants minimises secondary transcriptomic changes, enabling clearer identification and analysis of direct NMD targets as well as revealing UPF1 functions beyond canonical NMD. To dissect UPF1’s non-canonical functions, we generated helicase- and ATPase-deficient mutants in the eds1 background. While both are similarly impaired in NMD, they exhibit distinct growth phenotypes, with the ATPase mutant growing slower than the helicase mutant, suggesting additional non-NMD functional differences.UPF1-GFP localisation reveals that the protein is predominantly cytoplasmic but accumulates significantly in the nucleus following Leptomycin B treatment, which inhibits nuclear export. This observation supports active nucleo-cytoplasmic shuttling of UPF1 and implicates potential nuclear functions beyond its established cytoplasmic role. Given UPF1’s nuclear localisation, genome instability signatures, and growth defects in mutants, we hypothesised that UPF1 may function in R-loop metabolism. Indeed, our strand-specific Rloop profiling (ssDRIP-seq) in upf1 eds1 uncovers significant R-loop accumulation along gene bodies, suggesting that UPF1 might suppress co-transcriptional R-loops during elongation. Together, these findings reveal a novel nuclear function of UPF1 in R-loop homeostasis, distinct from its canonical NMD activity. The upf1 eds1 background provides a powerful genetic system to dissect UPF1’s essential and multifaceted roles in RNA biogenesis, without confounding influences from immune activation.