Nuclear myosin I acts in concert with polymeric actin to drive RNA polymerase I transcription

Abstract

Actin is associated with all three nuclear RNA polymerases and acts in concert with nuclear myosin I (NM1) to drive transcription. Practically nothing is known regarding the state of actin and the functional interplay of actin and NM1 in transcription. Here we show that actin and NM1 act in concert to promote RNA polymerase I (Pol I) transcription. Drugs that prevent actin polymerization or inhibit myosin function inhibit Pol I transcription in vivo and in vitro. Mutants that stabilize the polymeric state actin are tightly associated with Pol I and activate transcription, whereas a polymerization-deficient mutant does not bind to Pol I and does not promote rDNA transcription. Consistent with nuclear actin and myosin synergizing in transcription activation, NM1 mutants that lack specific functions, such as binding to ATP, actin, or calmodulin, are incapable of associating with Pol I and rDNA. The results show that actin polymerization and the motor function of NM1 are required for association with the Pol I transcription machinery and transcription activation. These observations provide insights into the cooperative action of actin and myosin in the nucleus and reveal an actomyosin-based mechanism in transcription.

Figure 1.

Inhibition of actin polymerization decreases Pol I transcription. (A) Drugs that inhibit actin polymerization impair Pol I transcription in vivo. RNA was isolated from HEK293T cells treated for 2 h with dimethylsulphoxide (DMSO), phalloidin (Pha., 1 μM), jasplakinolide (Jasp., 1 μM), cytochalasin D (Cyto.D, 2 μM), or latrunculin B (Lat.B, 2 μM). Pre-rRNA synthesis was measured by RT–qPCR using GAPDH mRNA as internal control. (B) Cytochalasin D and latrunculin B inhibit Pol I transcription in vitro. Nuclear extracts were preincubated for 30 min at 30°C with DMSO, phalloidin (Pha.), jasplakinolide (Jasp.), cytochalasin D (Cyto.D), or latrunculin B (Lat.B) before transcription was started. (C) Latrunculin B abolishes actin-mediated rescue of Pol I transcription. Nuclear extract was preincubated for 30 min either with buffer (lanes 1,2) or with 1 μg of anti-actin (Ac74) antibody (lanes 3–5) before recombinant Flag-tagged actin (5 μg) were added. The assay in lane 5 contained 10 μM latrunculin B (Lat.B). (D) Cofilin inhibits Pol I transcription in vitro. (Top panel) Transcription assays were conducted after preincubation of nuclear extracts for 30 min at 30°C with the indicated amounts of purified profilin or cofilin. The Coomassie-stained polyacrylamide gel in the middle panel shows the amounts of added profilin and cofilin. (Bottom panel) To assay the effect of profilin and cofilin on actin polymerization, FM3A cell lysate was incubated with increasing amounts of profilin or cofilin, and the level of F-actin was monitored by ultracentrifugation and Western blot analysis of pelleted actin using Ac74 antibody.

Figure 2.

Actin polymerization is required for association with the transcription machinery and activation of Pol I transcription. (A) Polymerization-deficient actin does not rescue Pol I transcription in actin-depleted nuclear extract. (Lanes 2–12) We added 1.2 or 2.5 μg of immunopurified Flag-tagged wild-type actin and the indicated point mutants to nuclear extracts that have been preincubated with 0.5 μg of anti-actin antibody (Ac74). In lane 1, the transcriptional activity of undepleted nuclear extract is shown. The capability of the mutants to polymerize or stabilize F-actin is indicated above. (B) Polymeric actin is associated with the Pol I transcription machinery. Nuclear extracts from HEK293T cells overexpressing Flag-tagged wild-type or mutant actin were incubated with immobilized anti-TIF-IA or anti-Pol I antibodies. Precipitated proteins were visualized on Western blots using antibodies against RPA116, TIF-IA, NM1, or the Flag epitope. (C) Mutants that stabilize F-actin stimulate Pol I transcription. RNA was isolated from HEK293T cells overexpressing wild-type or mutant Flag-tagged actin and pre-rRNA synthesis was monitored by RT–qPCR using GAPDH mRNA as internal control. Error bars represent standard deviation of three independent experiments. The expression levels of wild-type or mutant Flag-tagged actin were monitored on immunoblots using antibody against the Flag epitope (Supplemental Fig. S1). (D) Actin polymerization augments binding of Pol I to rDNA. Data from ChIP experiments showing rDNA occupancy of Pol I in HEK293T cells overexpressing wild-type actin or mutants R62D and S14C. Error bars represent standard deviation of three independent experiments.

Figure 3.

Actin and NM1 cooperate in Pol I transcription activation. (A) Both actin and NM1 are required to rescue transcription in nuclear extract treated with anti-actin antibody. Nuclear extract was preincubated for 20 min with buffer (lane 1) or 0.5 μg of anti-actin (Ac74) antibody (lanes 2–8), before the indicated amounts of recombinant actin or/and NM1 were added and transcription was started. (B) The association of actin and NM1 with Pol I is regulated by the ATP/ADP cycle. Nuclear extract from HEK293T cells expressing V5-tagged NM1 was incubated with immobilized anti-TIF-IA or anti-Pol I antibodies in the presence of 1 mM ATP, ADP, or ATP-γS. Precipitated proteins were visualized on Western blots using antibodies against RPA116, TIF-IA, the V5 epitope, or β-actin. (C) BDM inhibits rDNA transcription in vivo. RNA was isolated from HEK293T cells that have been treated with DMSO or BDM (2 mM, 12 h), and pre-rRNA synthesis was monitored by RT–qPCR. (D) BDM inhibits rDNA transcription in vitro. Nuclear extract was preincubated with DMSO or the indicated amounts of BDM (30 min at 30°C) before transcription was started, and run-off transcripts were visualized by autoradiography.

Figure 4.

The motor function is required for NM1 binding to Pol I and rDNA occupancy of the Pol I transcription machinery. (A) Scheme depicting the structure of NM1. The N-terminal domain is colored violet, the head is colored pink, the neck including the IQ motifs is colored yellow, and the positively charged tail is colored green. The dark-blue boxes mark regions involved in ATP or actin binding. (B) The association of NM1 with Pol I requires the motor function of NM1. Nuclear extract from HEK293T cells overexpressing wild-type or mutant V5-tagged NM1 was incubated with immobilized anti-TIF-IA or anti-Pol I antibodies, and precipitated proteins were visualized on Western blots using antibodies against RPA116, TIF-IA, actin, or the V5 epitope. (C) The motor function of myosin is required for the association of Pol I with rDNA. HEK293T cells expressing wild-type or mutant NM1-V5 under the control of a tetracycline-inducible promoter were treated with doxycycline for 12–16 h. The expression level of wild-type and mutant NM1-V5 was assayed on immunoblots using anti-V5 antibody (Supplemental Fig. S3). rDNA occupancy of Pol I was analyzed by ChIP using anti-RPA116 antibodies. Error bars represent standard deviation of three independent experiments.

Figure 5.

The actin-dependent motor function is required for NM1 binding to rDNA. (A) Actin and NM1 are associated with the pre-rRNA coding region and the intergenic spacer. Cross-linked chromatin from HEK293T cells stably expressing V5-tagged wild-type NM1 was precipitated with antibodies against Pol I (anti-RPA116), TIF-IA, actin (Ac40), or the V5 epitope, and immunoprecipitated DNA was analyzed by qPCR with the indicated primer pairs. Error bars represent standard deviation of three independent experiments. The scheme above outlines a representative mammalian rDNA repeat, illustrating the positions of the upstream terminator T₀, the rDNA promoter, the pre-rRNA coding region, the downstream terminators (T_1–10), and the intergenic spacer (IGS). (B) The motor function of NM1 is required for association with rDNA. HEK293T cells expressing V5-tagged NM1 or the indicated mutants under the control of a tetracycline-inducible promoter were treated with doxycycline for 12–16 h, and rDNA occupancy of NM1 was analyzed by ChIP using anti-V5 antibody. Error bars represent standard deviation of three independent experiments. The expression level of wild-type and mutant NM1-V5 was assayed by immunoblotting using anti-V5 antibody (Supplemental Fig. S3).

Figure 6.

The association of actin and NM1 with rDNA does not depend on ongoing transcription. (A) Actin and NM1 are associated with active and silent rRNA genes. Cross-linked chromatin from HEK293T cells expressing V5-tagged wild-type NM1 was precipitated with antibodies against TIF-IA, Pol I (anti-RPA116), TIP5, NM1 (anti-V5), and actin (Ac40). Precipitated DNA was either mock-digested or digested with HpaII, and the relative level of HpaII-resistant, inactive rDNA copies (dark bars), and unmethylated, active copies (light bars) was determined by real-time PCR using a primer pair that amplifies rDNA sequences from −150 to +32. (B) Inhibition of Pol I transcription does not affect rDNA occupancy of actin and NM1. HEK293 cells were treated for 1 h with 50 ng/mL actinomycin D (Act.D). (Left panel) Inhibition of pre-rRNA synthesis was monitored by RT–qPCR. The bar diagram at the right shows data from ChIP experiments comparing rDNA occupancy of Pol I, actin, and NM1 in DMSO-treated cells (light bars) and actinomycin D-treated cells (dark bars). Error bars represent standard deviation of three independent experiments.