HOS1 regulates Argonaute1 by promoting transcription of the microRNA gene MIR168b in Arabidopsis

Abstract

Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA-induced silencing complex have been well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that High Expression Of Osmotically Responsive Genes1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the Argonaute1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold-stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene Flowering Locus C (FLC). In a genetic screen for enhancers of sic-1, we isolated a loss-of-function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1-7 mutant flowered early and was smaller in stature than the wild-type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri-MIR168b and pre-MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.

Keywords: Arabidopsis thaliana; HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1; MIR168b; gene expression; microRNA; transcriptional regulation.

Figure 1

Identification and characterization of the hos1-7sic-1 mutant. (A) Diagram of the HOS1 gene and the hos1 mutant alleles used in this study. (B) Bioluminescence images of wild type (Col-0 ecotype (with gl-1 mutantion) harboring proRD29A-LUC transgene), sic-1, and hos1-7sic-1 under control and cold conditions, and the quantification of LUC intensity in wild type, sic-1, and hos1-7sic-1 DATA are average of 100 seedlings intensity, error bars represent s.d.’s (n = 100). (C) Northern analysis of transcript levels of transgene LUC and COR15A in gl-LUC, sic-1, and hos1-7sic-1. (D) qRT-PCR of the relative expression of LUC and RD29A in gl-LUC, sic-1, and hos1-7sic-1. Actin2 was used as internal control and error bars represent s.d.’s (n = 4).

Figure 2

HOS1 is required for proper accumulation of mature miR168a/b. (A) miRNA accumulation in hos1 mutants. Two hos1 mutant alleles (hos1-1 and hos1-3) showed a reduction in mature miR168a/b, while other miRNAs were not affected by the hos1 mutations. hos1-1 in C24 ecotype harboring proRD29A-LUC transgene and hos1-3 is T-DNA insertion mutant (Salk_069312c). U6 indicates RNA loading control. Signal intensity was measured with ImageJ and normalized to loading control U6. Relative expression was normalized to wild type. (B) Restored mature miR168a/b in complemented transgenic lines. The genomic DNA sequence of HOS1 with its native promoter was transformed into the hos1-3 mutant. miR168a/b was restored to wild type levels in the complemented transgenic lines. U6 indicates RNA loading control. Signal intensity was measured with ImageJ and normalized to loading control U6. Relative expression was normalized to wild type. (C) GFP-ICE protein detection by Western blot analysis. Wild type and hos1 mutant harboring over-expression of GFP-ICE1 were treated with 50 μM MG132 for 24 h to inhibit the proteasome mediated protein degradation. DMSO was used as the treatment control. The increase in GFP-ICE1 protein level in the wild type under MG132 treatment compared to DMSO control treatment indicates the expected inhibition of the proteasome degradation pathway. Coomassie blue staining of Rubisco was used as loading control. (D) Accumulation of miR168a/b after MG132 treatment. MG132 treatment did not affect the accumulation of mature miR168a/b, indicating that the accumulation of miR168a/b was not affected by HOS1-mediated ubiquitination and degradation of ICE1. Signal intensity was measured with ImageJ and normalized to loading control tRNA. Relative expression normalized to wild type.

Figure 3

HOS1 regulates the transcription of MIR168b. (A) Reduction of pri-MIR168b in hos1 mutants. pri-MIR59a, pri-MIR168a, and pri-MIR168b levels were examined in hos1-1 and hos1-3 mutants. Actin2 was used as internal control and error bar represents s.d.’s (n = 4). (B) Reduction of pre-MIR168b in hos1 mutants as detected by Northern analysis. U6 was used as an internal control. Signal intensity was measured with ImageJ and normalized to loading control U6. Relative expression was normalized to wild type. (C) ChIP analysis showing the decrease in wild type and hos1-1 at MIR168a (left panel). The ChIP signal was normalized by actin2 and error bars represent s.d.’s (n = 4).

Figure 4

HOS1 associates with the chromatin at the promoter region of MIR168b. ChIP assays using proHOS1::HOS1-4xmyc transgenic plants. ChIP assays were carried out using an anti-MYC antibody. FLC P1 and P2 were used as positive controls as reported previously (Jung et al., 2013). ChIP signal was normalized by actin2 and error bars represent s.d.’s (n = 4)

Figure 5

HOS1 is required for proper maintenance of AGO1. (A) Increase in AGO1 transcript in hos1 mutants. qRT-PCR (upper panel) and Northern analysis (lower panel) revealed an increase in AGO1 mRNA in hos1-1 and hos1-3 alleles compared to their respective wild types. Actin2 was used as internal control and error bars represents s.d.’s (n = 4) for qRT-PCR. Northern signal was measured with imageJ and normalized to loading control actin2. Relative expression was normalized to wild type. (B) Increase in AGO1 protein level in hos1 mutants. A Coomassie blue-stained band corresponding to the Rubisco largest subunit is shown as a loading control.