Periodic Reporting for period 1 - LoAGOding (Stay home or go beyond: deciphering the mechanisms underlying ARGONAUTE1 loading and sRNA movement in plants)
Período documentado: 2024-01-01 hasta 2025-12-31
Small RNAs (sRNAs) are powerful regulators of gene expression and are essential for development, stress resilience and antiviral defence in plants. When loaded into an ARGONAUTE (AGO) protein, they act as a guide for AGO to silence a target gene. A central open challenge is that AGO1, the main sRNA effector in plants, operates across compartments: it is found in the cytoplasm, where it executes post-transcriptional gene silencing, and in the nucleus, where it loads micro RNAs (miRNAs, a type of sRNA), and it is associated with additional regulatory functions. Nonetheless, we still lack a clear mechanistic understanding of where other sRNAs are loaded into AGO1, how nuclear and cytoplasmic “loading environments” compete, and how this partitioning shapes sRNA mobility between cells.
This project set out to establish a compartment-resolved view of AGO1 loading by: (i) dissecting sRNA loading when AGO1 is confined to the nucleus versus the cytoplasm, (ii) decipher the connection between miRNA biogenesis (D-bodies) and AGO1 loading complexes, and (iii) testing how the subcellular site of loading influences local and systemic sRNA activity.
This project set out to establish a compartment-resolved view of AGO1 loading by: (i) dissecting sRNA loading when AGO1 is confined to the nucleus versus the cytoplasm, (ii) decipher the connection between miRNA biogenesis (D-bodies) and AGO1 loading complexes, and (iii) testing how the subcellular site of loading influences local and systemic sRNA activity.
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.
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.
This project moves beyond correlative localization studies by delivering causal, compartment-resolved principles for AGO1 function. It establishes that:
• Cytoplasmic AGO1 alone is sufficient to sustain effective sRNA-mediated regulation, capturing both phenotypic rescue and transcriptome-scale correction.
• AGO1 loading is competitive and compartment-biased: nuclear loading can outcompete cytoplasmic loading for a substantial fraction of sRNAs, extending competition to sRNAs generated from nuclear transcripts.
• A transferable interaction-imaging framework is now established to map the architecture and assembly rules of nuclear small-RNA hubs.
• DCL1-HYL1 and HYL1-AGO1 interactions promotes colocalization and pinpointed a specific HYL1 domain as a regulatory node in nucleo-cytoplasmic trafficking.
• HST-mediated export and AGO1-associated routes act in a complementary manner, such that blocking both severely compromises downstream developmental programs.
Together, these advances position AGO1 trafficking not as a secondary detail, but as a central control layer coordinating distinct silencing outcomes.
• Cytoplasmic AGO1 alone is sufficient to sustain effective sRNA-mediated regulation, capturing both phenotypic rescue and transcriptome-scale correction.
• AGO1 loading is competitive and compartment-biased: nuclear loading can outcompete cytoplasmic loading for a substantial fraction of sRNAs, extending competition to sRNAs generated from nuclear transcripts.
• A transferable interaction-imaging framework is now established to map the architecture and assembly rules of nuclear small-RNA hubs.
• DCL1-HYL1 and HYL1-AGO1 interactions promotes colocalization and pinpointed a specific HYL1 domain as a regulatory node in nucleo-cytoplasmic trafficking.
• HST-mediated export and AGO1-associated routes act in a complementary manner, such that blocking both severely compromises downstream developmental programs.
Together, these advances position AGO1 trafficking not as a secondary detail, but as a central control layer coordinating distinct silencing outcomes.