Uncovering a novel function of the CCR4-NOT complex in phytochrome A-mediated light signalling in plants

Phytochromes are photoreceptors regulating growth and development in plants. Using the model plant Arabidopsis, we identified a novel signalling pathway downstream of the far-red light-sensing phytochrome, phyA, that depends on the highly conserved CCR4-NOT complex. CCR4-NOT is integral to RNA metabolism in yeast and animals, but its function in plants is largely unknown. NOT9B, an Arabidopsis homologue of human CNOT9, is a component of the CCR4-NOT complex, and acts as negative regulator of phyA-specific light signalling when bound to NOT1, the scaffold protein of the complex. Light-activated phyA interacts with and displaces NOT9B from NOT1, suggesting a potential mechanism for light signalling through CCR4-NOT. ARGONAUTE 1 and proteins involved in splicing associate with NOT9B and we show that NOT9B is required for specific phyA-dependent alternative splicing events. Furthermore, association with nuclear localised ARGONAUTE 1 raises the possibility that NOT9B and CCR4-NOT are involved in phyA-modulated gene expression.

Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.