To uncouple nuclear and cytoplasmic functions, Arabidopsis lines expressing wild-type AGO1 (wtAGO1), a nuclear-restricted AGO1 (nucAGO1), or a cytoplasmic-restricted AGO1 (cytAGO1) were analysed in ago1 mutant backgrounds. The project combined developmental phenotyping with transcriptomics (RNA-seq), small-RNA profiling (sRNA-seq), and direct measurement of AGO1-bound small RNAs (AGO1 immunoprecipitation followed by sRNA-seq).
A key achievement is the demonstration that cytAGO1 is sufficient to support canonical miRNA-mediated gene regulation in vivo: it restored wild-type-like development and rescued misregulated miRNA targets, with only minimal transcriptomic differences compared to wtAGO1. In contrast, nucAGO1 only partially restored target regulation and produced a strong phenotype on specific classes of small RNAs that were broadly reduced, often even below mutant levels. Crucially, the project provides direct evidence that AGO1 loading is compartment-biased and competitive. nucAGO1 efficiently loads certain small RNAs but is nearly depleted of others, whereas cytAGO1 loads sRNAs robustly. In plants co-expressing nucAGO1 and cytAGO1 variants with distinct tags, nucAGO1 reduced sRNA loading into cytAGO1, supporting a quantitative advantage for nuclear loading, potentially due to proximity to sRNA biogenesis.
In parallel, the project established an enabling platform to disentangle nuclear processing and loading hubs. A multicolour FRET-FLIM imaging strategy was designed to distinguish D-body interactions from AGO1-associated loading complexes within the same nuclear context. In vivo assays provided functional clues that DCL1-HYL1 and HYL1-AGO1 interactions promotes accumulation and colocalization and pinpointed a specific HYL1 domain as a regulatory node in nucleo-cytoplasmic trafficking. Stable Arabidopsis reporter lines under native promoters were initiated to transfer these analyses to endogenous settings.
Finally, to connect sRNA loading with its cell-to-cell mobility, genetic materials combining compartment-restricted AGO1 with the miRNA pathway factor HASTY (HST) were generated. Loss of HST together with compartment restriction produced strong developmental failure and sterility, indicating that AGO1 routing and AGO-independent export act as complementary pathways. Initial mobility reporter observations are consistent with nuclear retention of AGO1 suppressing a non-cell-autonomous silencing phenotype, while cytoplasmic AGO1 preserves it.
