In plants, small RNAs (sRNA) are loaded into ARGONAUTE (AGO) proteins to fulfill their regulatory functions. Micro RNAs (miRNAs), one of the most abundant classes of endogenous sRNAs, are preferentially loaded into AGO1. Such loading, long believed to happen exclusively in the cytoplasm, was recently proposed to also occur in the nucleus. Here we identified CARP9, a nuclear-localized intrinsically disordered protein, as a factor promoting miRNA activity in Arabidopsis thaliana. Mutations in the CARP9-encoding gene led to a milder reduction of miRNAs levels, an impaired gene silencing, and characteristic morphological defects. Intriguingly, we found that CARP9 was able to interact with HYL1, but not with other proteins of the miRNA biogenesis machinery. In the same way, CARP9 appeared to interact with mature miRNA, but not with pri-miRNA, positioning it after miRNA processing in the miRNA pathway. We also found that CARP9 was able to interact with AGO1 promoting its interaction with HYL1 to facilitate the loading of miRNAs in AGO1. Plants deficient in CARP9 displayed reduced levels of AGO1-loaded miRNAs, partial retention of miRNA in the nucleus, and reduced levels of AGO1. Collectively, our data suggest that CARP9 might modulate HYL1-AGO1 crosstalk acting as a scaffold, stabilizing AGO1 and allowing the proper loading of miRNAs in the effector complex.
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