Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis

Abstract

MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1-HYL1-SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA-HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.

Fig. 1.

tgh-1 reduces the accumulation of miRNAs and siRNAs. (A) The accumulation of miRNAs and miR172* in three genotypes. (B) The accumulation of siRNAs in three genotypes. For miR159/319: Upper band, miR159; lower band, miR319 (34). The numbers indicate the relative abundance of small RNAs among the three genotypes and represent the mean of three repeats (P < 0.05). U6 blot was used as a loading control. Col-0, WT control for tgh-1; tgh-1+TGH, tgh-1 harboring TGH genomic DNA. (C) Deep sequencing analysis of miRNAs in tgh-1 and WT. Small RNA libraries were generated from inflorescences. The miRNA abundance was calculated as reads per million, and a log₂-transformed ratio of tgh-1/Wt was plotted. Each circle represents one miRNA. Thick lines indicate median values.

Fig. 2.

tgh-1 impairs multiple DCL activities. (A) Increased pri-miRNA levels in inflorescences of tgh-1. The levels of pri-miRNAs in tgh-1 were normalized to those of UBIQUITIN 5 and compared with Col. Error bars indicate SD of three technical replications (*P < 0.05 and **P < 0.01). (B) Reduced production of siRNAs from dsRNAs in the tgh-1 protein extracts. (C) Reduced production of 21- and 24-nt siRNA in the tgh-1 protein extracts. Numbers below indicate siRNA production in tgh-1 relative to control. (D) Quantification of overall siRNA production in tgh-1 extracts relative to the control extracts. Data are presented as mean and SD (n = 7; ***P < 0.001). (E) The pri-miR162b processing in tgh-1, dcl1-9, and WT. The reaction was stopped after 120 min. Numbers indicate overall miRNA production in tgh-1 and dcl1-9 extracts relative to their respective control extracts and represent the mean of three experiments (P < 0.05).

Fig. 3.

TGH associates with the DCL1 complex. (A) MBP-TGH pulls down DCL1-YFP. (B) DCL1-YFP pulls down MBP-TGH. (C) MBP-TGH pulls down HYL1 and SE. Protein precipitates were analyzed by Western blot by using anti-MBP, anti-GFP, and anti-HYL1 antibodies, respectively. One percent input proteins were used for MBP-TGH and MBP, and 2% input proteins were used for YFP, DCL1-YFP, and HYL1. (D) BiFC analysis between TGH and the components of DCL1 complex. TGH and SE were fused with cCFP whereas DCL1, HYL1, SE, and AGO1 were fused with nVenus. Respective pair of cCFP and nVenus fusion proteins was coinfiltrated into leaves, and fluorescence signals were examined ∼40 h after coinfiltration. The interaction of paired proteins will result in yellow fluorescence (green in image). More than 30 nuclei were examined for each pair, and a graph is shown. DNA was stained with DAPI to visualize the nuclei (blue).

Fig. 4.

TGH is an RNA-binding protein. (A) TGH-MBP and MBP proteins used in the in vitro RNA binding assay. The proteins were resolved on an SDS-polyacrylamide gel and detected by Coomassie brilliant blue staining. (B) TGH binds pri-miR162b and premiR162b in vitro. (C) TGH binds pri-miRNA in vivo. RIP was performed with the anti-HA antibody. C, Col-0; T, tgh-1 harboring a TGH::TGH-HA transgene. One eighth immunoprecipitates was analyzed by Western blot. Input protein was 2%. No RT was performed with the pri-miR167a primers. Input RNA was 5%. (D) TGH binds pre-miRNAs in vivo. RT was performed with primer P2. The first-round PCR was done with primers P1 and P2. The Second round PCR was performed with primers P3 and P4. Primer P4 recognizes the junction between adaptor and premiRNA. Open box, adaptor; light/dark gray box, miRNA/miRNA*; black box, region between miRNA and miRNA*.

Fig. 5.

TGH contributes to in vivo HYL1–pri-miRNA interaction. (A) Detection of HYL1 protein after immunoprecipitation. Immunoprecipitation was performed with the anti-HYL1 antibody. (B) and (C) association between HYL1 and pri-miR171a and pri-miR167a was impaired in tgh-1. C, Col-0; t, tgh-1. One eighth immunoprecipitates were analyzed by Western blot. Input protein was 2% of total input proteins. The amount of pri-miR167a and pri-miR171a was determined by qRT-PCR and normalized to the input. AtSN1B was used as a negative control (*P < 0.05 and **P < 0.01).