Reduced <i>DUO1</i> and CYCB1 expression in <i>arid1-1</i>.

<p>(A) Expression of sperm-specific genes in mature pollen of wild type and <i>arid1-1</i>. Error bars represent the SE from the mean of three biological replicates. (B) Expression of DUO1-RFP in wild type and <i>arid1-1</i>. Representative images for each genotype were acquired with the same exposure times. White and red arrows indicate reduced DUO1-RFP signal in bicellular pollen and mature pollen, respectively. Scale bar, 10 µm. (C) Expression of CYCB1-GFP in the bicellular pollen of wild type and <i>arid1-1</i>, respectively. The red arrows indicate visible GFP accumulation of CYCB1 in the generative nucleus of wild type pollen, and the white arrows indicate unchanged GFP signal in the vegetative nuclei of the <i>arid1-1</i> pollen. Scale bar, 10 µm.</p

ARID1 physically associates with Histone Deacetylase 8 (HDA8).

<p>(A) Yeast cells co-expressing the indicated plasmids were grown on control (without Tryptophan and Leucine) or high stringency selection (without Tryptophan, Leucine, Histidine, and with 10 mM 3-AT) medium. <i>ARID1</i>, full length cDNA of <i>ARID1</i>; <i>ARID1-C</i>, the C-terminus-containing ELM2 domain of ARID1; <i>HDA8</i>, full length cDNA of <i>HDA8</i>. AD is pGAD10. The cultures from each of the indicated strains were diluted 100-fold and spotted. Three colonies were streaked for each pair. (B) ARID1 interacts with HDA8 by GST pulldown assay. Whole cell extracts from wild type or ARID1-GFP plants were applied onto GST and GST-HDA8 (abbreviated GST-H8) beads. GFP and Hsc70 (loading control) antibodies were used in immunoblotting. (C) Stained protein gel showing proteins used for the GST pulldown assay. 1/30 of the amount of each protein used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004421#pgen-1004421-g005" target="_blank">Figure 5A</a> was resolved by SDS-PAGE. GST-H8, full length HDA8 fused to GST; GST alone was used as a control. GST-H8 is marked with an arrowhead. (D) ARID1 interacts with HDA8 by Co-IP. Inflorescences from ARID1-Myc; HDA8-YFP (abbreviated H8-YFP) doubly transgenic plants or from wild type were immunoprecipitated with Myc antibody. Myc, GFP and Hsc70 (loading control) antibodies were used for immunoblotting.</p

Altered histone acetylation in <i>arid1-1</i>.

<p>(A) Expanded pattern of histone acetylation signal in <i>arid1-1</i> by immunofluorescence analysis. Mature pollen from wild type and <i>arid1-1</i> plants was incubated with anti-H3 (control) and anti-H3K9ac antibodies. Representative examples for <i>arid1-1</i> show histone acetylation in the vegetative nucleus (indicated by white arrowheads). 50–100 pollen grains for each genotype were examined. (B) Reduced histone acetylation at the <i>DUO1</i> promoter in the <i>arid1-1</i> mutant by ChIP. Inflorescences from wild type and <i>arid1-1</i> were used for a ChIP assay with anti-H3, anti-H3K9ac, and anti-H3K4me3 antibodies. ChIP-DNA was used for PCR by amplifying 35 cycles for both <i>EIF4A1</i> (negative control) and the <i>DUO1_3</i> fragment, which bound ARID1. Similar results were obtained from three independent biological replicates; the results shown are from one replicate.</p

ARID1 binds to <i>DUO1</i>.

<p>(A) Schematic of subfragments of <i>DUO1</i> genomic DNA. The black rectangles represent exons. The black triangle indicates the region where ARID1 was most abundant. The position of the ATG was set to 1, and the fragments upstream or downstream were numbered; for example fragment “1” is −1321 to −1019 bp. (B) ChIP performed with wild type (gray bars) or <i>ARID1-GFP</i> (black bars) with GFP antibody (upper panel) and No antibody control (lower panel). <i>EIF4A1</i> was used an internal negative control. The results were reproducible in two biological replicates. Error bars show SD calculated from three technical replicates. (C) DNA binding assay. Proteins were resolved by SDS-PAGE and then immunoblotted using anti-ARID1. The input lanes have 1/5 of the amount of ARID1 protein used in the DNA binding assay. The lanes numbered 3, 4, 5 and 10 (corresponding to regions mentioned in (A)) have 1/6 of the DNA-bound protein.</p

Specific expression of <i>ARID1</i> in pollen and disruption of <i>ARID1</i> results in defective sperm cell formation.

<p>(A) Expression of <i>ARID1</i> by RT-PCR. Ro, roots; Se, seedlings; Le, leaves; Cf, closed flower buds; Of, open flowers; Pi, unpollinated pistils; Po, mature pollen; Si, siliques. The RT (−) control PCR was performed with <i>UBQ5</i> primers. (B) Representative siliques of WT and <i>arid1-1</i> and complementation test. Undeveloped ovules are indicated with arrows. (C) Percentage of normal seeds (dark grey), aborted seeds (lighter grey), and undeveloped ovules (lightest grey) from self-pollinated plants are shown. Error bars represent standard deviation from the mean. (D) Seed set analysis in antisense <i>ARID1</i> transgenic plants. Numbers in the bottom row represent individual T1 lines, and the corresponding numbers in the top row indicate the percentage of reduced seed set in each line. The gel shows the expression of <i>ARID1</i> as assessed by RT-PCR analysis. (E) Distribution of unicellular microspores (UM, lightest grey), bicellular pollen (BP, lighter grey), and tricellular pollen (TP, dark grey) in mature anthers. At least 600 pollen grains were stained with DAPI and used for statistical analysis; Error bars represent standard deviation from the mean.</p

An ARID Domain-Containing Protein within Nuclear Bodies Is Required for Sperm Cell Formation in <i>Arabidopsis thaliana</i>

<div><p>In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and <i>DUO1</i> (<i>DUO POLLEN 1</i>), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how <i>DUO1</i> itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (<u>A</u>T-<u>R</u>ich <u>I</u>nteracting <u>D</u>omain)-containing protein, ARID1, that is specifically required for sperm cell formation in <i>Arabidopsis</i>. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of <i>DUO1</i> activation. An <i>arid1</i> mutant and antisense <i>arid1</i> plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of <i>DUO1</i>. In vitro and in vivo evidence showed that ARID1 binds to the <i>DUO1</i> promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the <i>arid1</i> mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on <i>DUO1</i> through association with histone acetylation.</p></div

Model for ARID1 function during sperm cell formation in <i>Arabidopsis</i>.

<p>miR159 plays a major role in restricting <i>DUO1</i> expression in the vegetative cell. As pollen development proceeds, miR159 abundance is gradually decreased and ARID1 expands its expression into the generative cell, possibly by responding to the decreased repressive role of miR159 in bicellular pollen. ARID1 then promotes <i>DUO1</i> activation by directly binding to the <i>DUO1</i> promoter, and thereby facilitates the initiation of sperm cell formation. On the other hand, ARID1 might repress expression of unknown negative regulators (orange) of cell cycle progression, by altering the epigenetic status of the generative cell. Once sperm cells are formed, ARID1 gradually decreases until it is eliminated from germline cells, so that DUO1 is steadily present and negative regulators possibly related to germline function start to be active, due to dissociation of ARID1 and histone deacetylase. Thus we hypothesize that the alteration of epigenetic status during sperm cell formation is correlated with a change in the subcellular localization of ARID1, which could facilitate cell cycle progression of the two consecutive mitoses. VN, vegetative nucleus; GN, generative cell; SC, sperm cells.</p

Reduced male transmission in <i>arid1-1</i> plants.

<p>Reduced male transmission in <i>arid1-1</i> plants.</p

Lariat RNAs competitively inhibit DCL1/HYL1 binding to pri-miRNAs.

<p>(A) Lariat RNAs associate with DCL1 using a RIP assay performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006422#pgen.1006422.g002" target="_blank">Fig 2B</a>. Immunoprecipitated RNAs were analyzed by qRT-PCR with divergent primers to detect the indicated lariat RNAs. <i>UBQ5</i> was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (B) Lariat RNAs associate with HYL1 using a RIP assay performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006422#pgen.1006422.g002" target="_blank">Fig 2C</a>. qRT-PCR was performed according to (A). <i>hyl1-2</i> was used as the negative control. <i>UBQ5</i> was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (C) R-EMSA to determine HYL1 binding to <i>pri-miR167b</i> in the presence of circular RNAs from Col-0 or <i>dbr1-2</i> plants. Recombinant MBP-HYL1-D1D2 (MBP-HYL1) was incubated with a 5’_biotin_labeled <i>pri-miR167b</i> probe after addition of different amounts of circular RNAs isolated from Col-0 or <i>dbr1</i>-2 inflorescences, respectively. The arrow indicates the HYL1-<i>pri-miR167b</i> complex. (D) Hybridization intensities were quantified and normalized to the controls (lane 2 in C), and are shown in the line graph. Bars represent the average normalized intensity of three biological replicates.</p

Intron Lariat RNA Inhibits MicroRNA Biogenesis by Sequestering the Dicing Complex in <i>Arabidopsis</i>

<div><p>Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null <i>dbr1</i> mutants in both animals and plants are embryo lethal, but the mechanism underlying the lethality remains unclear. Here we characterized a weak mutant allele of <i>DBR1</i> in <i>Arabidopsis</i>, <i>dbr1-2</i>, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The <i>dbr1-2</i> mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex <i>in vivo</i> and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in <i>dbr1-2</i>. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex <i>in vivo</i>, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.</p></div