Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

Abstract

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

Keywords: ARGONAUTE1; RNA processing; THO/TREX complex; cotranscription.

Fig. 1.

AGO1 genetically functions with FCA and FLD to repress FLC. (A) Co-IP in nuclear extracts from wild-type seedlings (Control) or seedlings expressing SDG26-TAP. IP: immunoprecipitation; IB: immunoblot. Asterisk indicates nonspecific signal. (B) Expression of spliced and unspliced FLC relative to UBC in Col-0 and ago1-25 seedlings. Data are normalized to wild-type Col-0. Data are presented as the mean ± SEM (n = 3). Asterisks indicate significant differences between the indicated plants (**P ≤ 0.007136, *P ≤ 0.031062, two-tailed t test). (C) Expression of spliced and unspliced FLC relative to UBC in various genotypes. Data are normalized to Col-0. Data are presented as the mean ± SEM (n = 3 to 5). Two-tailed P value from multiple t test corrected by Holm–Sidak method; ns, not significant. (D) Expression of spliced and unspliced FLC relative to UBC in Col FRI and ago1-25 FRI seedlings. Data are normalized to Col FRI. Data are presented as the mean ± SEM (n = 6). Asterisks indicate significant differences between the indicated plants (*P ≤ 0.029432, ****P ≤ 0.000551, two-tailed t test). (E) Expression of spliced and unspliced FLC relative to UBC in C2 and ago1-25 C2 seedlings. Data are normalized to C2. Data are presented as the mean ± SEM (n = 3). Asterisks indicate significant differences between the indicated plants (***P ≤ 0.002662, ****P ≤ 0.000588, two-tailed t test).

Fig. 2.

AGO1 associates with COOLAIR and influences COOLAIR processing. (A) Schematic diagram showing FLC gene structure and COOLAIR transcripts. Black and gray boxes represent FLC and COOLAIR exons, respectively. Black and gray lines represent FLC and COOLAIR introns, respectively. The arrow indicates the transcription start site (TSS). Short black lines with letters underneath indicate positions of amplicons in qPCR amplification. (B) RIP–qPCR analyzing HA-AGO1 enrichment on nascent COOLAIR transcript. Wild-type Col-0 was used as background control. The x axis corresponds to the fragments shown in A. Data are mean ± SD (n = 3). (C) The ratio of proximal-to-distal isoforms of COOLAIR transcripts (refer to the schematic in A) in various genotypes relative to Col-0. Data are mean ± SEM (n = 3). (D) The splicing efficiency of distal intron (spliced/unspliced) determined through chromatin-bound RNA analysis. Data are normalized to Col-0. Data are mean ± SD (n = 3).

Fig. 3.

AGO1 promotes COOLAIR R-loop removal and FLC chromatic silencing. (A) DRIP–qPCR determining R-loop level in Col-0 and ago1-25. The number on the x axis is the distance to FLC TSS. Data are percentage of input normalized to a region without COOLAIR transcript (between 6,000 and 7,000 base pairs to FLC TSS). Data are presented as mean ± SEM (n = 3). (B) ChIP analysis of H3K4me1 level at FLC in various genotypes. The number on the x axis is the distance to FLC TSS. Data are mean ± SD (n = 3).

Fig. 4.

The THO/TREX complex up-regulates FLC and promotes distal COOLAIR. (A) Expression of spliced and unspliced FLC relative to UBC in various genotypes. Data are normalized to Col-0. Data are presented as the mean ± SEM (n = 3). (B) Expression of spliced and unspliced FLC relative to UBC in various genotypes. Data are normalized to Col-0. Data are presented as the mean ± SEM (n = 3). (C) Expression of spliced and unspliced FLC relative to UBC in various genotypes. Data are normalized to Col FRI. Data are presented as the mean ± SEM (n = 3). (D) RIP-qPCR analyzing HRP1-GFP enrichment on FLC mRNA in transgenic line expressing HPR1-GFP. Transgenic line expressing GFP alone was used as negative control. The x axis refers to the fragments shown in Fig. 2A. Data are mean ± SD (n = 3). (E) RIP-qPCR analyzing HRP1-GFP enrichment on nascent COOLAIR. Transgenic line expressing GFP was used as negative control. The x axis is the distance to COOLAIR 5′. Data are mean ± SD (n = 3). (F) The ratio of proximal-to-distal isoforms of COOLAIR transcripts (refer to the schematic in Fig. 2A) in various genotypes relative to Col-0. Data are mean ± SEM (n = 3).

Fig. 5.

Model for how modulation of antisense RNA processing determines sense transcriptional output. Antagonistic assembly of cotranscriptional machinery with RNA Pol II influences transcript processing. During COOLAIR transcription, FCA, AGO1, and components of NTC/NTR link to RNA Pol II and promote proximal COOLAIR. THO/TREX dynamically competes for RNA Pol II to promote distal COOLAIR. The differential composition of the cotranscriptional regulators with the Pol II complex influences the chromatin state locally, which in turn influences the transcriptional output of the whole locus.