Targeted Recruitment of the Basal Transcriptional Machinery by LNK Clock Components Controls the Circadian Rhythms of Nascent RNAs in Arabidopsis

Plasmid Construction, Plant Material, and Growth Conditions

LNK1 and LNK3 plant overexpression vectors were generated by PCR-mediated amplification of the LNK1 and LNK3 coding sequences followed by cloning into the pENTR/D-TOPO vector (Invitrogen). The LNK1 and LNK3 coding sequences were subsequently cloned into the plant destination vector pGWB417/517 (Nakagawa et al., 2007a, 2007b) following the manufacturer’s recommendations (Invitrogen). RVE8 and domain vectors were generated by cloning RVE8-FL, ΔLCL (nucleotides 1 to 624), and the LCL domain (nucleotides 625 to 885) first into the pENTR/D-TOPO vector and then into the plant destination vector pGWB505 (Nakagawa et al., 2007a, 2007b).

Bacterial expression vectors were generated by cloning the coding sequences of LNK1, SSRP1, RNA Pol II CTD, RVE8-FL, ΔLCL, and LCL into the pENTR/D-TOPO vector. Each coding sequence was subsequently cloned into pDEST565 or pDEST_HIS_MBP (Nallamsetty et al., 2005) vectors using the LR reaction (Invitrogen). For MBP-LNK3, the coding sequence of LNK3 was amplified by PCR and cloned into the pET_HIS_MBP vector (Bogomolovas et al., 2009) after digestion with NcoI and XhoI restriction enzymes (Roche) and ligation with T4 DNA ligase (Roche).

Arabidopsis thaliana plants were transformed using Agrobacterium tumefaciens (GV2260)-mediated DNA transfer (Clough and Bent, 1998). Seedlings were stratified at 4°C in the dark for 3 d on Murashige and Skoog (MS) agar medium supplemented with 3% sucrose. Plates were transferred to LD (12 h of light/12 h of dark) with 60 to 100 μmol·quanta·m⁻²·s⁻¹ cool white fluorescent light at 22°C. The TOC1pro:LUC (Perales and Más, 2007), RVE8-FL-ox (Farinas and Mas, 2011), rve8 (SALK_016333C) (Farinas and Mas, 2011), lnk1 lnk2 (SALK_024353, GK_484F07) (Rugnone et al., 2013), rve4 rve6 rve8 (Hsu et al., 2013), and ssrp1-2 (SALK_001283) (Lolas et al., 2010) lines were described elsewhere. Full-length RVE8-ox, ΔLCL-ox, and LCL-ox were transformed into the wild-type background as well as the rve8 mutant background.

Protein Sequence Alignment and Phylogenetic Analyses

Multiple sequence alignment of the amino acid sequence of the LCL domain was performed using protein sequences from the EnsemblPlants database (http://plants.ensembl.org/index.html). The sequences were subjected to BLAST analysis against the LCL domain of Arabidopsis RVE8. Protein homologs with the closest sequence identity were aligned with ClustalW using the Bioedit program (Bioedit 7.2.5).

Phylogenetic analysis was performed using the maximum likelihood method based on a JTT matrix-based model (Jones et al., 1992). The tree with the highest log likelihood (−923.86) was selected. The initial tree for the heuristic search was obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model and then selecting the topology with superior log likelihood value. The number of bootstrap replicates was 1000. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). The analysis involved 45 amino acid sequences. All positions containing gaps and missing data were eliminated. There were 56 positions in the final data set. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

Analyses of Hypocotyl Growth, Flowering Time, and Confocal Imaging

Seeds were stratified on plates with MS agar medium without sucrose for 4 d in the dark at 4°C. Seeds were then exposed to white light (80 μmol·quanta·m⁻²·s⁻¹) for 4 h and kept in the dark for 20 h following exposure to constant red light (42 μmol·quanta·m⁻²·s⁻¹) for 7 d. Approximately 20 to 25 seedlings were used for hypocotyl length measurements using ImageJ software (National Center for Biotechnology Information [NCBI]). For flowering time assays, seeds were synchronized for 4 d at 4°C and placed on soil. Plants were grown under long-day conditions (16 h of light/8 h of dark) with a light intensity of 80 to 100 μmol·quanta·m⁻²·s⁻¹ in walk-in chambers (INKOA). Bolting time and the number of rosette leaves of 10 to 15 plants were counted when the inflorescence stems reached 1 cm high. Two-tailed t tests were performed for statistical analyses (*P < 0.05, **P < 0.01, and ***P < 0.001). Two biological replicates were performed with plants grown at different times. Confocal analyses were performed with plants grown on MS agar medium supplemented with 3% sucrose under long-day cycles. Fluorescence signals from hypocotyl and root cells were imaged using an Olympus Fluoview FV1000 confocal microscope with a 515-nm argon laser (excitation, 488 nm; emission, 510 nm).

In Vivo Luminescence Assays

Seven-day-old plants synchronized under LD cycles (12 h of light/12 h of dark) at 22°C were transferred to 96-well plates and resynchronized for an additional 24 h under LD cycles before switching to LL conditions. In vivo luminescence assays were performed as described previously (Takahashi et al., 2015) with an LB960 luminometer (Berthold Technologies) using Microwin software (Mikrotek Laborsysteme). Periods, phases, and amplitudes were estimated using the Fast Fourier Transform-Non‐Linear Least Squares suite using the BioDare online tool (www.biodare.ed.ac.uk) (Zielinski et al., 2014). Two-tailed t tests were performed for statistical analyses (*P < 0.05, **P < 0.01, and ***P < 0.001). For inhibitor analyses, different concentrations of Flap (1, 5, and 10 µM) and Selic (10, 100, and 500 µM) were vacuum-infiltrated for 5 to 8 min into 7-d-old seedlings before luminescence analyses. Two biological replicates were performed with seedlings grown at different times. Each biological replicate included 8 to 12 seedlings per condition and/or genotype.

Gene Expression Analyses by RT-qPCR

Seedlings were synchronized under LD cycles for 7 d and subsequently transferred to LL for 2 d. Samples were taken every 4 h over the third day under LL. RNA was purified using a Maxwell 16 LEV simplyRNA tissue kit following the manufacturer’s recommendations (Promega). Purified RNA was treated with RNase-free Turbo DNase (Ambion), and single-stranded cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad) with 1 μg of RNA. qPCR was performed with iTag Universal SYBR Green Supermix (Bio-Rad) or Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) with a 96-well CFX96 Touch Real-Time PCR detection system (Bio-Rad). The expression data were normalized to IPP2 (ISOPENTENYL PYROPHOSPHATE: DIMETHYL-ALLYL PYROPHSPHATE ISOMERASE) using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). For inhibitor analyses, Selic (500 µM) was vacuum-infiltrated for 5 to 8 min into 7-d-old seedlings, and samples were harvested the following day at different circadian times, as indicated. The expression data were normalized to 18S rRNA using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Two biological replicates were performed with seedlings grown and sampled at different times. Two to three technical replicates were performed within the same biological replicate. Primers used in this study are listed in Supplemental Table 1.

Yeast-Two Hybrid Screening

The coding sequence of the LCL domain (nucleotides 625 to 825) was PCR amplified and fused to the C-terminal end of LexA in the pB27 vector. The construct was used as a bait to screen a random-primed cDNA library of Arabidopsis constructed into the pP6 vector (Hybrigenics Services). Using a mating approach with YHGX13 and L40ΔGal4 strains, ∼64 million clones were screened and around 322 His+ colonies were selected on a medium lacking histidine, tryptophan, and leucine. The prey fragments of these positive clones were sequenced and used to search the GenBank database (NCBI) to identify potential interacting proteins with the LCL domain.

Protein Expression, Purification, and in Vitro Pull-Down

Transformed Escherichia coli cells (BL21, DE3) were grown until the OD₆₀₀ values reached 0.6 to 0.8. Isopropyl β-D-1-thiogalactopyranosidemediated induction of MBP-LNK3, GST-CTD, and GST-SSRP1 was performed at 28°C for 4 to 6 h. MBP-LNK1, GST-ΔLCL, and GST-LCL were induced at 17°C overnight. Bacteria were lysed by sonication for 2 to 3 min (30 s on, 30 s off, high intensity) using a sonicator (Bioruptor, Diagnode). Recombinant proteins were purified using gravity flow columns with amylose resin for MBP fusion proteins (New England Biolabs) and Glutathione Sepharose 4B for GST-tagged proteins (GE Healthcare). The purified recombinant proteins were concentrated using Amicon centrifugal filters (EMD Millipore).

For in vitro pull-down assays between ΔLCL, LCL, and LNKs, proteins were incubated in pull-down binding buffer (1× PBS, pH 7.4, 0.2% glycerol, 0.6% Triton X-100, and 1 mM β-mercaptoethanol) for 2 h at 4°C with end-over-end rotation. Amylose resin was then added and incubated for another 2 h at 4°C with end-over-end rotation. Beads were washed four to six times with pull-down washing buffer (1× PBS, pH 7.4, 0.6% Triton X-100, and 1 mM β-mercaptoethanol), and protein complexes were released by heating at 95°C for 5 min in 2× SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol). Protein samples were analyzed by immunoblotting using an anti-MBP antibody (reference sc-809, Santa Cruz Biotechnology) (1:1000 dilution) or a monoclonal anti-GST antibody (reference 8-326; Thermo Fisher Scientific) (1:1000 dilution).

To test the interaction between LNKs and RNA Pol II CTD or SSRP1, proteins were processed as described above with minor modifications. Briefly, proteins were incubated in pull-down binding buffer (20 mM Tris-HCl, pH 7.9, 20% glycerol, 1 mM EDTA, 5 mM MgCl₂, 0.1% Nonidet P-40, 1 mM DTT, 0.2 mM PMSF, and 0.1 M NaCl) for 2 h at 4°C with end-over-end rotation. Glutathione Sepharose 4B was then added and incubated for another 2 h at 4°C with end-over-end rotation. Beads were washed four times with pull-down washing buffer (20 mM Tris-HCl, pH 7.9, 20% glycerol, 1 mM EDTA, 5 mM MgCl₂, 0.1% Nonidet P-40, 1 mM DTT, 0.2 mM PMSF, and 0.1 M NaCl), and protein complexes were released by heating at 95°C for 5 min in 2× SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol). Protein samples were analyzed by immunoblotting with an anti-MBP antibody (reference sc-809, Santa Cruz Biotechnology) (1:1000 dilution) or with a monoclonal anti-GST antibody (reference 8-326, Thermo Fisher Scientific) (1:1000 dilution). Two biological replicates were performed per experiment and/or condition.

In Vivo Co-IP Assays in Plants

Analyses of LNKs and LCL interactions were performed with 10-d-old seedlings grown under LD cycles. At least two biological replicates with seedlings grown and sampled at different times were performed. Around 1 g of seedlings was ground in liquid nitrogen and resuspended in 1 mL of co-IP binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.4% Nonidet P-40, 1 mM EDTA, 2 mM DTT, 5% glycerol, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132). Protein concentration was measured using standard curves with the Bradford method (Bradford, 1976). Supernatants containing equal amounts of total protein were incubated with 25 μL of GFP-trap magnetic beads (Chromotek) for 1 to 2 h at 4°C with end-over-end rotation. Beads were washed with co-IP washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132). The protein complexes were released from the beads with 50 μL of 2× SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol) and heating at 95°C for 5 min. The co-IP samples were then analyzed by immunoblot analyses using an anti-GFP antibody (reference A11122, Invitrogen) (1:2500 dilution) and an anti-Myc antibody (reference M4439, Sigma-Aldrich) (1:2500 dilution).

Analyses of the interaction of LNKs with RNA Pol II or SSRP1 were performed following the same protocol described above with slight modifications. Briefly, samples were resuspended in 1 mL of co-IP binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.4% Nonidet P-40, 5 mM MgSO₄, 2 mM CaCl₂, 2 mM DTT, 5% glycerol, 1 mM PMSF, 30 μg/mL leupeptin, 30 μg/mL aprotinin, 30 μg/mL E-64, 7.5 μg/mL antipain, 3 μg/mL pepstatin, 7.5 μg/mL chymostatin, and 50 μM MG-132). Protein concentration was measured using standard curves with the Bradford method (Bradford, 1976). Supernatants containing equal amounts of total protein were treated with DNase I (Promega) or Benzonase (Novagen). Samples were then incubated with 25 μL of Myc-trap agarose beads (Chromotek) for 1 to 2 h at 4°C with end-over-end rotation. Beads were washed with co-IP binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.4% Nonidet P-40, 5 mM MgSO₄, 2 mM CaCl₂, 2 mM DTT, 5% glycerol, 1 mM PMSF, 30 μg/mL leupeptin, 30 μg/mL aprotinin, 30 μg/mL E-64, 7.5 μg/mL antipain, 3 μg/mL pepstatin, 7.5 μg/mL chymostatin, and 50 μM MG-132). Protein complexes were released from the beads using 50 μL of 2× SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol) and heating at 95°C for 5 min. The co-IP samples were analyzed by immunoblot analysis using a monoclonal anti-Myc antibody (reference M4439, Sigma-Aldrich) (1:2500 dilution), an anti-RNA Pol II CTD repeat YSPTSPS (phospho S2) antibody (reference ab5095, Abcam) (1:1000 dilution), an anti-RNA Pol II CTD repeat YSPTSPS (phospho S5) antibody (reference ab5131, Abcam) (1:1000 dilution), an anti-RNA Pol II antibody (reference at-300, Santa Cruz Biotechnology) (1:1000 dilution), and an anti-SSRP1 antibody (1:1000 dilution) (Duroux et al., 2004). For inhibitor analyses, Selic (300 µM) was vacuum-infiltrated for 8 min into 10-d-old seedlings at ZT3, and samples were harvested at ZT7 the following day. For Ponceau S staining, the membrane was incubated with Ponceau S solution (0.1% Ponceau S and 1% acetic acid) for 5 min with shaking and washed twice by water before imaging.

ChIP and Sequential ChIP Assays

Plants were grown under LD cycles for 10 to 14 d, and samples were collected under LD conditions or the third day under LL at the indicated time points. At least two biological replicates with seedlings grown and sampled at different times were performed. ChIP assays were essentially performed as described previously (Perales and Más, 2007) with some modifications. Briefly, 1 to 2 g of seedlings was fixed in fixation buffer (10 mM Tris-HCl, pH 8.0, 0.4 M sucrose, 1 mM EDTA, 1 mM PMSF, 0.25% Triton X-100, and 1% formaldehyde) for 10 to 15 min under a vacuum. The fixation was stopped by adding glycine to a final concentration of 0.125 M and vacuum for 10 min. Seedlings were washed twice with 50 mL of cold water, frozen in liquid nitrogen, and stored at −80°C. Samples were ground in liquid nitrogen to a fine powder and resuspended in extraction buffer I (10 mM Tris-HCl, pH 8.0, 0.4 M sucrose, 5 mM EDTA, 5 mM β-mercaptoethanol, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132). Following filtration with Miracloth (EMD Millipore), the cells were centrifuged at 1000g for 20 min and washed with extraction buffer II (10 mM Tris-HCl, pH 8.0, 0.25 M sucrose, 10 mM MgCl₂, 1% Triton X-100, 5 mM EDTA, 5 mM β-mercaptoethanol, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132) to purify nuclei. The nuclei were then resuspended in 1 mL of nuclei lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132) and sonicated 4 min (30 s on, 30 s off, low intensity) with a sonicator (Bioruptor, Diagnode). Nuclear debris was removed by centrifugation at maximum speed. Approximately 20 to 25 μg of chromatin was then diluted with 1 mL of ChIP dilution buffer (15 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1 mM EDTA, 1.1% Triton X-100, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132) and incubated with the corresponding antibody overnight at 4°C. Protein-DNA complexes were collected by incubation with 50 μL of equilibrated Protein G beads (Dynabeads, Invitrogen) for 2 to 3 h. The beads were sequentially washed with low-salt buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132), high-salt buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132), LiCl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132), and TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Protein-DNA complexes were released by incubation with 300 μL of ChIP elution buffer for 1 h at 65°C. Reverse cross-linking was performed by adding 0.2 M NaCl and incubating at 65°C overnight. DNA was purified using a Gel Extraction Kit (Qiagen) following the manufacturer’s recommendation. qPCR was performed with iTag Universal SYBR Green Supermix (Bio-Rad) or Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) using a 96-well CFX96 Touch Real-Time PCR detection system (Bio-Rad). For inhibitor analyses, Flap (10 µM) or Selic (300 µM) was vacuum-infiltrated for 8 min into 12-d-old seedlings at ZT3, and samples were harvested at ZT7 the following day. To take into account differences in the immunoprecipitation efficiency in the different samples, ACT7 was used as a positive control in ChIP assays of the basal transcriptional machinery. For ChIP analyses with LNKs or RVE8 domains, it is obviously not possible to use ACT7 as a positive control. A two-tailed t test was performed for statistical analysis (*P < 0.05, **P < 0.01, and ***P < 0.001).

Sequential ChIP assays (Xie and Grotewold, 2008) were performed following the same ChIP procedure described above but including 2× washes with low-salt buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL E-64, 5 μg/mL antipain, 1 μg/mL pepstatin, 5 μg/mL chymostatin, and 50 μM MG-132) and 2× washes with TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) after the first round of immunoprecipitation with the anti-MYC antibody (reference M4439, Sigma-Aldrich) (1:500 dilution). Complexes were eluted with 15 mM DTT and incubated at 37°C for 30 min. Chromatin was then diluted in Sequential ChIP buffer (15 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 5 µg/mL leupeptin, 1 µg/mL aprotinin, 5 µg/mL antipain, 1 µg/mL pepstatin, 5 µg/mL chymostatin, and 50 µm MG132) and concentrated using Amicon centrifugal filters (EMD Millipore). Samples were then incubated overnight at 4°C either with an anti-Myc antibody (reference M4439, Sigma-Aldrich) (1:500 dilution) or with an anti-SSRP1 antibody (Duroux et al., 2004) (1:200 dilution). Complexes were washed, eluted, purified, and amplified as described above.

Nascent RNAs

Nascent RNA analyses were performed as described previously (Hetzel et al., 2016) with minor modifications. Briefly, seedlings were grown in MS medium supplemented with 3% sucrose for 5 d under LD cycles at 22°C. Plates were then moved to LL conditions, and ∼20-g samples were collected at the indicated time points on the third day under LL. At least two biological replicates with seedlings grown and sampled at different times were performed. Samples were homogenized with an Ultra Turrax T-25 homogenizer in 100 mL of ice-cold grinding buffer (300 mM sucrose, 20 mM Tris, pH 8.0, 5 mM MgCl₂, 5 mM KCl, 0.2% [v/v] Triton X-100, 5 mM β-mercaptoethanol, and 35% [v/v] glycerol) at 4°C. Samples were then filtered through Miracloth (EMD Millipore) before being passed through a 60-µm cell strainer into 50-mL Falcon tubes. Tubes were centrifuged at 5000g for 10 min at 4°C. Pellets were then washed twice by homogenization with 30 mL of cold grinding buffer using a microstreaker. Pellets were resuspended in 2 mL of freezing buffer (50 mM Tris, pH 8.0, 5 mM MgCl₂, 20% [v/v] glycerol, and 5 mM β-mercaptoethanol), and nuclei were counted under a light microscope using a Neubauer chamber. Nuclei were then divided into aliquots in 1.5-mL tubes and frozen in liquid N₂. Nuclear run-on of ∼5 to 6 × 10⁶ nuclei was performed by adding 200 μL of 3× nuclear run-on reaction buffer (15 mM Tris-HCl, pH 8.0, 450 mM KCl, 7.5 mM MgCl₂, 1.5% [v/v] 20% sarkosyl, 1.5 mM DTT, 0.2 units/μL SUPERase-in [Fisher Scientific], 375 µM ATP, 375 µM GTP, 60 nM CTP, and 375 µM BrUTP [Sigma-Aldrich]). After 5 min of incubation at room temperature, run-on was stopped by adding 140 μL of DNase Mix (15 μL of 10× RQ1 DNase I buffer, 50 μL of nuclease-free deionized water, and 5 μL of RQ1 DNase [Promega]) for 15 min at room temperature, followed by the addition of 160 μL of STOP Mix (20 mM EDTA, 200 mM NaCl, 1% [v/v] SDS, and 0.3 mg/mL glycogen) plus 20 μL of 2.5 mg/mL proteinase K and incubation for 30 min at 37°C. Nuclei were centrifuged for 10 min at 1000g at 4°C, and RNA was extracted using a TRIzol-based protocol in which 250 μL of high-salt buffer (0.8 M sodium citrate [NaH₂(C₃H₅O(COO)₃)] and 1.2 M NaCl) and 1 μL of RNase-free glycogen were added for isopropanol precipitation. Prior to BrUTP precipitation, BrdU antibody beads (sc-32323 AC; Santa Cruz Biotechnology) were washed twice with ice-cold GRO binding buffer (0.25× saline-sodium-phosphate-EDTA buffer, 0.05% [v/v] Tween, 37.5 mM NaCl, and 1 mM EDTA). Approximately 50 μL of each RNA sample was diluted with 450 μL of cold GRO binding buffer and incubated with 40 μL of equilibrated BrdU antibody beads. The reaction was incubated under slow rotation for 1.5 h at 4°C. Beads were spun down for 20 min at 1000g at 4°C. Beads were then resuspended in 200 μL of cold grinding buffer and transferred to a Millipore MC column (UFC30HVNB EMD Millipore). After a spin down for 1 min at 1000g, beads were washed twice with 500 μL of cold grinding buffer for 5 min under fast rotation at 4°C. Columns were then moved to fresh 1.5-mL tubes, and RNA was eluted with 200 μL of TRIzol LS under gentle shaking for 5 min at room temperature. After a second TRIzol elution, 100 μL of nuclease-free deionized water was added to the column and eluted. RNA was extracted using a TRIzol-based protocol following the manufacturer’s recommendations. cDNA synthesis was performed as described previously (Roberts et al., 2015). A High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used for reverse transcriptase-mediated PCR. RT-qPCR analyses were performed in a CFX96 Touch Real-Time PCR detection system using Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent Technologies). The primers used for nascent RNA detection span exon-intron boundaries in order to exclude the amplification of mature mRNA, which was also verified by the use of primers amplifying exon-exon boundaries. Nascent RNA primers for ACT7 and UBQ5 were used as controls. The expression data were analyzed using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). The primers used are listed in Supplemental Table 1.

Accession Numbers

Sequence data in this study can be found in the Arabidopsis Genome Initiative under the following accession numbers: RVE8 (AT3G09600), LNK1 (AT5G64170), LNK2 (AT3G54500), LNK3 (AT3G12320), LNK4 (AT5G06980), SSRP1 (AT3G28730), SPT16 (AT4G10710), RNA Pol II (AT4G35800), TOC1 (AT5G61380), PRR5 (AT5G24470), ACT7 (AT5G09810), UBQ5 (AT3G62250), PPR (AT5G55840), IPP2 (AT3G02780), and TA3 (AT1G37110).

Supplemental Data

• Supplemental Figure 1. Characterization of ΔLCL-ox.

• Supplemental Figure 2. Multiple Sequence Alignment of the LCL Domain Amino Acid Sequence in Plants.

• Supplemental Figure 3. Phylogenetic Analysis of the Closest Homologs to RVE8.

• Supplemental Figure 4. Characterization of LCL-ox.

• Supplemental Figure 5. Direct Interaction of LNKs with the LCL Domain of RVE8 and Total RNA Pol II Accumulation at the PRR5 and TOC1 Loci.

• Supplemental Figure 6. In Vitro Interaction of LNK1 with RNA Pol II and Binding of LNK1 and RVE8 to the PRR5 and TOC1 Loci.

• Supplemental Figure 7. LNKs and SSRP1 Are Important for RNA Pol II Recruitment at Core Clock Loci.

• Supplemental Figure 8. Analyses of RNA Pol II S5P and S2P Inhibition.

• Supplemental Figure 9. Role of LNKs in the Regulation of PRR5 and TOC1 Transcript Synthesis.

• Supplemental Table 1. Primers Used in This Study.