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

Abstract

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.

Figure 1.

Characterization of carp9 mutants. A, Gene structure of CARP9 showing single nucleotide deletion of the carp9-1 allele and T-DNA insertion sites in carp9-2, carp9-3, carp9-4, and carp9-5. Black boxes and lines represent exons and introns, respectively; gray boxes represent coding sequence, while blue boxed shows 5′ and 3′ UTR regions. A white asterisk marks the position of the stop codon that results from the frameshift caused by the carp9-1 single nucleotide deletion (G2464Del). B, Phenotypic characterization of carp9 mutants, control lines (reporter and Col-0), and carp9-1 mutants complemented by the overexpression of the CARP9 cDNA (carp9-1; Pro^35S:CARP9). Twenty-one-day-old plants, fully expanded siliques, and 40-d-old plants are displayed. Bars = 1 cm. Plants were imaged individually, digitally extracted, and mounted on a single black background panel to facilitate comparison and observation. C, Analysis of the flowering time of control, carp9 mutant, and complemented lines grown in long-day photoperiod as measured by the number of rosette leaves or the number of days to bolting. Error bars show means ± se (n ≥ 15). D, Bioluminescence activity, quantified by a luminometer, of 12 leave discs belonging to 20-d-old carp9-1 mutants and reporter plants. Error bars show means ± se (n ≥ 12). Asterisks indicate significance by two-tailed, unpaired t test Holm-Sidak corrected (*P < 0.05). E, Bioluminescence activity as measured with a CCD camera, in 20-d-old carp9-1 mutants and reporter plants. Luminescence intensity is color scaled from low (blue) to high (white). Two pots containing 12 plants each were imaged individually, digitally extracted, mounted in a single black background panel, and displayed in the figure. F, RNA blots for detecting amiRLuc. U6 was used as a loading control. G, Phenotypic features of 18-d-old carp9 mutants, control lines, and carp9-1/carp9-2 compound heterozygous mutants. Bar = 1 cm. Plants were imaged individually, digitally extracted, and mounted in a single black background panel to facilitate comparison and observation.

Figure 2.

CARP9 mutants present impaired miRNA activity. A, RNA blots for detecting endogenous miRNAs. U6 was used as a loading control. The relative abundance of each miRNA, indicated above each band, was calculated by measuring the band intensity using ImageJ and relativized to the corresponding control plant (reporter for carp9-1 and complemented plants, and Col-0 for carp9-2). B, miRNA levels, as measured by RT-qPCR, in mutants and control lines. Error bars show means ± 2× se (n ≥ 4). Asterisks indicate significance by two-tailed, unpaired FDR-corrected t test (*P < 0.05) were considered significant. C, Mean expression levels of individual miRNAs in carp9-1 and hyl1-2 plants relative to Col-0 plants. Horizontal segments indicate the median of the expression levels. Each dot corresponds to a single miRNA or collapsed miRNA family. Dark and light dots show differentially (false discovery rate adjusted P-value < 0.05) and not differentially accumulated miRNAs, respectively. D, Expression of miRNA targets in control and mutant plants as measured by RT-qPCR. Error bars show means ± 2× se (n ≥ 4). Asterisks indicate significance by two-tailed, unpaired t test Holm-Sidak corrected (*P < 0.05).

Figure 3.

Conservation analysis of CARP9 across the plant kingdom. A, Top, gene structure of CARP9 as shown in Figure 1A. Bottom, the CARP9 protein structure; in purple is marked a putative NSL signal and its amino acid sequence. The occluding/ELL-like domain is marked in red. B, Top and middle, disordered score, and regions in AtCARP9 amino acid sequence according to MobiDB (Piovesan et al., 2018). Bottom, amino acid alignment quality of CARP9-like genes using Jalview software (Waterhouse et al., 2009); positions are based on AtCARP9 full sequence. C, Phylogeny of CARP9-like genes in embryophytes. The unrooted consensus tree was generated using the maximum likelihood method. Colors represent different lineages of plant species, referenced in the figure. AtCARP9 is highlighted with a black arrow. Supplemental Figure S3B shows the fully annotated tree. D, Confocal microscopy images showing the nuclear localization of eGFP- and mCherry-tagged versions of CARP9 in Nicotiana benthamiana transiently transformed leaves (left) and stably transformed Arabidopsis plants (right). Scale bars = 5 μm.

Figure 4.

CARP9 interacts with HYL1 and mature miRNAs, but not with the miRNA processing machinery. A to C, Expression of pri-miRNA, pri-artificial miRLUC, HYL1, and SE in control and mutant plants as measured by RT-qPCR. D, HYL1 and SE quantification by immunoblot in samples extracted from carp9-2 and reporter plants. The detection of ACTIN was used as a loading control. E, ChIP experiment using either anti-GFP, anti-DCL1, or anti-IgG antibodies in plants that express a GFP-tagged version of CARP9 to detect MIRNAs loci associated with the proteins. Primers used for the amplification are listed in the Supplemental Table S3 and based on a previous report (Fang et al., 2015). ACTIN gene was used as control not targeted by CARP9 nor DCL1. F, Confocal microscopy images simultaneously showing the localization of CARP9 with DCL1, SE, HYL1, and CPL1 in transiently transformed N. benthamiana leaves. Scale bars = 5 μm. G, BiFC assay in N. benthamiana cells showing CARP9 interaction with HYL1 and se. Negative interactions are displayed in a wider magnification to show the negative interactions better. Positive interactions in a wider magnification are shown in Supplemental Figure S4. Scale bars = 5 μm. H, Interaction of CARP9 with HYL1, but not with se, as detected by yeast two-hybrid assays. GAL4 activation domain (AD); GAL4 DNA binding domain (BD); −LT, medium without Leu and Trp; −LTH, selective medium without Leu, Trp, and His. Each column shows a 1:10 serial dilution. I, CARP9-HYL1 interaction detected by co-IP assays. Leaves of Arabidopsis plants transformed with Pro-35S::CARP9-eGFP were immunoprecipitated using an anti-GFP antibody. Interacting HYL1 was identified using an antibody targeting the endogenous protein. J and K, pri-miRNAs (J) and mature miRNAs or miRNAs passender strands (miRNA*s; K) associated with CARP9, or HYL1, as quantified by RIP-RT-qPCR in samples extracted from plants expressing a GFP-tagged version of CARP9 and immunoprecipitated with either an anti-GFP, anti-HYL1, or anti-IgG antibodies. Values are given as a percentage to the qPCR signal detected in the input samples. L, Precisely processed miRNA reads at all highly expressed MIRNA loci. Each dot represents an individual miRNA; horizontal black bars indicate medians. miRNA levels in all samples are expressed as a ratio to the precisely processed miRNAs in Col-0 plants grown at 23°C in long-day (LD) photoperiod. In A to C, E, and J to K, error bars show means ± 2× se (n ≥ 4). Asterisks indicate significance by two-tailed, unpaired t test (*P < 0.05 and **P < 0.01).

Figure 5.

CARP9 interacts with AGO1 to modulate its nuclear miRNA loading. A, BiFC assay in N. benthamiana cells showing CAP9 interaction with AGO1 and with HYL1 as controls. The nuclear transcription factors PIF4 and TCP15 were used as negative controls and displayed in a wider magnification. Scale bars = 5 μm. B, Co-IP assays. Protein samples extracted from Col-0 wild-type plants or plants transformed with a 35S::CARP9-eGFP were immunoprecipitated using an anti-GFP antibody, AGO1-CARP9 interaction was then detected using an anti-AGO1 antibody. C, Interaction of CARP9 with HSP90 as detected by yeast two-hybrid assays. GAL4 activation domain (AD); GAL4 DNA binding domain (BD); −LT, medium without Leu and Trp; −LTH, selective medium without Leu, Trp, and His. Each column shows a 1:10 serial dilution. D, Co-IP assays. Protein samples extracted from Col-0 wild-type or carp9-2 plants were immunoprecipitated using an anti-AGO1 antibody, AGO1-HYL1 interaction was then detected using an anti-HYL1 antibody. AGO1 was detected to test IP efficiency. E, Relative amount of mature miRNA bound to AGO1 as measured by stem-loop RT-qPCR of samples immunoprecipitated using an anti-AGO1 antibody. Co-IPed miRNAs were normalized to the levels of the same miRNA in the input samples. For both mutants, the miRNA levels were then expressed as relative to their corresponding control. Error bars show means ± 2× se (n = 4). Asterisks indicate significance by two-tailed, unpaired t test (*P < 0.05 and **P < 0.01). No-antibody samples and ago1-36 mutant plants were used as negative controls for the IP experiment, not showing detectable signal in the assayed conditions. F, RNA blots for detecting miRNAs in different cell fractions. Quantification of U6 and tRNAs were used as a loading control and to monitor the purity of the nuclear/cytoplasmic fractions. G, Quantification of the miRNA distribution measured in F. Band intensity was quantified by ImageJ and normalized by the corresponding loading control. Distributions of miRNAs in the nuclear/cytoplasmic fractions were then expressed as relative to Col-0 (marked as a dashed line). H, Quantification of the miRNA distribution in nucleus versus cytoplasm fractions as measured by RT-qPCR. Each dot represents an independent replicate. Significant differences were tested with an ANOVA test: between miRNAs (P = 0.00484 and between genotypes, P= 0.00013).

Figure 6.

AGO1 stability is compromised in CARP9 mutants. A, AGO1 levels quantified by immunoblot in samples extracted from carp9 mutants and control plants. Levels of ACTIN were measured as a loading control. B and C, Expression of pri-miRNA168 (B) and AGO1 (C) as measured by RT-qPCR. AGO1 transcript levels were measured using two sets of primers; a pair amplifying the 3′ end of the transcript (Primers A) and a pair flanking the miR168 recognition site in the AGO1 mRNA (Primers B). Error bars show means ± 2× se (n = 4. Asterisks indicate significance by two-tailed, unpaired t test (*P < 0.05). D, AGO1 levels, as measured by immunoblots, in cytoplasmic or nuclear cell fractionated samples. ACTIN and Histone 3 (H3) were used to verify the purity of the fractions. E, Immunoblot quantification of AGO1 levels in mutant and control plants treated with the proteasome inhibitor MG132.

Figure 7.

A model for the role of CARP9 as a scaffold in a post-pri-miRNA processing and nuclear AGO1 loading complex. After the processing of pri-miRNAs by DCL1, the mature duplex remains associated with HYL1 and SE, which later is replaced by HEN1. CARP9 is recruited to this postprocessing complex by its binding to HYL1. Nuclear AGO1 is then associated with the complex and HSP90 interacting with CARP9, a process leading to the enhanced AGO1 stability and loading of AGO1 with the mature miRNAs. Loaded AGO1 is then exported to the cytosol to silence their target genes.