The Intrinsically Disordered Protein CARP9 Bridges HYL1 to AGO1 in the Nucleus to Promote MicroRNA Activity

Abstract

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 ARGONAUTE1 (AGO1). Such loading, long believed to happen exclusively in the cytoplasm, was recently proposed to also occur in the nucleus. Here we identified CONSTITUTIVE ALTERATIONS IN THE SMALL RNAS PATHWAYS9 (CARP9), a nuclear-localized, intrinsically disordered protein, as a factor promoting miRNA activity in Arabidopsis (Arabidopsis thaliana). Mutations in the CARP9-encoding gene led to a mild reduction of miRNAs levels, impaired gene silencing, and characteristic morphological defects, including young leaf serration and altered flowering time. Intriguingly, we found that CARP9 was able to interact with HYPONASTIC LEAVES1 (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. CARP9 was also able to interact with AGO1, promoting its interaction with HYL1 to facilitate miRNA loading 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 HYL1AGO1 crosstalk, acting as a scaffold for the formation of a nuclear post pri-miRNA processing complex that includes at least HYL1, AGO1 and HSP90. In such a complex, CARP9 stabilizes AGO1 and mature miRNAs, allowing the proper loading of miRNAs in the effector complex.