In plants, small RNAs are loaded into ARGONAUTE (AGO) proteins to fulfill their regulatory functions. MicroRNAs (miRNAs), one of the most abundant classes of endogenous small RNAs, 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 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 primary 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 HYL1-AGO1 cross talk, acting as a scaffold for the formation of a nuclear post-primary miRNA-processing complex that includes at least HYL1, AGO1, and HEAT SHOCK PROTEIN 90. In such a complex, CARP9 stabilizes AGO1 and mature miRNAs, allowing the proper loading of miRNAs in the effector complex.

在植物中，小 RNA 被加载到 ARGONAUTE (AGO) 蛋白中以实现其调节功能。MicroRNA (miRNA) 是最丰富的内源小 RNA 类别之一，优先加载到 AGO1 中。这种负载长期以来被认为只发生在细胞质中，最近被认为也发生在细胞核中。在这里，我们确定了小 RNA 通路的组成型改变9 (CARP9)，一种核定位的、本质上无序的蛋白质，是拟南芥 (Arabidopsis thaliana ) 中促进 miRNA 活性的一个因素。CARP9 编码基因的突变导致 miRNA 水平轻度降低、基因沉默受损和特征性形态缺陷，包括幼叶锯齿和开花时间改变。有趣的是，我们发现 CARP9 能够与 HYPONASTIC LEAVES1 (HYL1) 相互作用，但不能与 miRNA 生物发生机制的其他蛋白质相互作用。以同样的方式，CARP9 似乎与成熟 miRNA 相互作用，但不与初级 miRNA 相互作用，将其定位在 miRNA 通路中的 miRNA 加工之后。CARP9 还能够与 AGO1 相互作用，促进其与 HYL1 的相互作用，从而促进 AGO1 中的 miRNA 加载。缺乏 CARP9 的植物表现出负载 AGO1 的 miRNA 水平降低、细胞核中 miRNA 部分保留以及 AGO1 水平降低。总的来说，我们的数据表明 CARP9 可能调节 HYL1-AGO1 串扰，充当形成核后初级 miRNA 处理复合物的支架，该复合物至少包括 HYL1、AGO1 和热休克蛋白 90。在这样的复合物中, CARP9 稳定 AGO1 和成熟 miRNA，从而允许效应复合物中正确加载 miRNA。