Ars2 promotes proper replication-dependent histone mRNA 3' end formation

Abstract

Ars2 is a component of the nuclear cap-binding complex that contributes to microRNA biogenesis and is required for cellular proliferation. Here, we expand on the repertoire of Ars2-dependent microRNAs and determine that Ars2 regulates a number of mRNAs, the largest defined subset of which code for histones. Histone mRNAs are unique among mammalian mRNAs because they are not normally polyadenylated but, rather, are cleaved following a 3' stem loop. A significant reduction in correctly processed histone mRNAs was observed following Ars2 depletion, concurrent with an increase in polyadenylated histone transcripts. Furthermore, Ars2 physically associated with histone mRNAs and the noncoding RNA 7SK. Knockdown of 7SK led to an enhanced ratio of cleaved to polyadenylated histone transcripts, an effect dependent on Ars2. Together, the data demonstrate that Ars2 contributes to histone mRNA 3' end formation and expression and these functional properties of Ars2 are negatively regulated by interaction with 7SK RNA.

Figure 1. Ars2 regulates a subset of microRNAs

(A) HeLa cells were transfected with control siRNA or 3 siRNAs targeted to either Ars2 (Ars2-1, Ars2-2, Ars2-3) or DGCR8 (DGCR8-1, DGCR8-2, DGCR8-3). Protein was harvested three days following transfection to confirm specific depletion of Ars2 or DGCR8 by Western blot. (B) HeLa cells were transfected with the siRNAs as in (A) and RNA was isolated three days later and analyzed separately on Affymetrix GeneChip® miRNA Arrays. The number of microRNAs decreased at least 2-fold (log2) following transfection of all three siRNAs per gene are depicted by Venn Diagram. (C) Bar graphs showing 27 microRNAs determined by microarray to decrease 2-fold (log2) or more following depletion of DGCR8 or Ars2. Bars represent the average of the three siRNAs targeting DGCR8 or Ars2 shown in (A). (D) Bar graphs showing 6 microRNAs determined by microarray to decrease 2-fold (log2 ) or more following depletion of Ars2 but not DGCR8. Bars represent the average of the three siRNAs targeting DGCR8 or Ars2 shown in (A). (E) HeLa cells were transfected with two siRNAs targeted to Ars2 (Ars2-1, Ars2-2) or a control siRNA (ctl). Three days later RNA was isolated and TaqMan® microRNA assays were used to detect the mature microRNAs indicated. Bars represent relative quantification using the ΔΔC_t method +/− 95% confidence interval of three replicates. U6 snRNA was used as an endogenous control. (F) HeLa cells were transfected with siRNA to Ars2 (Ars2-2) or a control siRNA (ctl) and three days later RNA was isolated. Precursor and mature microRNAs indicated were detected by Northern blot. Ethidium bromide staining of 5S rRNA and tRNAs are shown as loading controls. (G) RNA from (E) was reverse transcribed and TaqMan®-based qPCR was performed for the indicated primary microRNA transcripts. Bars represent relative quantification using the ΔΔC_t method +/− 95% confidence interval of three replicates. U6 snRNA was used as an endogenous control.

Figure 2. Ars2 regulates expression of numerous mRNAs and associates with replication-dependent histone mRNAs

(A) RNA used for the miRNA array in Figure 1b was reverse-transcribed and hybridized to Affymetrix GeneChip® Human Gene 1.0ST Arrays. The number of mRNAs decreased at least 2-fold (log2) following transfection of all three siRNAs targeting Ars2 or DGCR8 are depicted by Venn Diagram. (B) The number of mRNAs increased at least 2-fold (log2) following transfection of all three siRNAs Ars2 or DGCR8 are depicted by Venn Diagram. (C) To confirm microarray results, HeLa cells were transfected independently with control siRNAs (c1, c2, c3) or three siRNAs to Ars2 (a1, a2, a3) and the indicated mRNA transcript levels were measured by TaqMan®-based qPCR. Bars represent relative quantification using the ΔΔC_t method normalized to c1 +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (D) To determine if Ars2-containing protein complexes could associate with genes found to increase after Ars2 depletion, paraformaldehyde crosslinking followed by immunoprecipitation (PFA-CLIP) with a monoclonal antibody to Ars2 (2G10) or control antibody (SP/0) was performed. RNA isolated from the precipitates was reverse transcribed with random hexamer primers and used for qPCR with TaqMan® primer/probe sets to the indicated genes. Bars represent relative quantification using the ΔΔC_t method normalized to control antibody CLIP +/- 95% confidence interval of three replicates. Non-specifically bound 18S rRNA was used as an endogenous control.

Figure 3. Ars2 binds to and regulates the expression and processing of replication-dependent histone transcripts

(A) To determine if Ars2 could directly associate with replication-dependent histone mRNAs, ultraviolet light crosslinking followed by immunoprecipitation (UV-CLIP) with a monoclonal antibody to Ars2 (9A12) or control antibody (SP/0) was performed. RNA isolated from the precipitates was reverse transcribed with random hexamer primers and used for qPCR with TaqMan® primer/probe sets to the indicated genes. Bars represent relative quantification using the ΔΔC_t method normalized to control antibody CLIP +/− 95% confidence interval of three replicates. Non-specifically bound 18S rRNA was used as an endogenous control. (B) HeLa cells were transfected with siRNA to Ars2 (Ars2-3) or a control siRNA and 3 days later RNA was isolated. cDNA synthesis was performed using either oligo(dT) or random hexamer primers. Transcript levels were measured by TaqMan®-based qPCR using the assays outlined in the inset table. Because the TaqMan® assays used for HIST2H2BE and HIST2H2AG amplified a region downstream of their 3′ cleavage sites they only detected non-cleaved transcripts (i.e. those that extend beyond the normal 3′ cleavage sites), whereas the assays for HIST1H3H and HIST3H2A were upstream of their 3′ cleavage sites and could therefore differentiate between polyadenylated transcripts (dT) and total mRNA expression (HEX). Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (C) Top - Schematic of the human HIST2H2BE gene with scale in kilobases (kb). The arrows indicate the position of primers used for PCR analysis. Bottom - HeLa cells were transfected with siRNAs to Ars2 (Ars2-1, Ars2-2) or control siRNAs and three days later RNA was isolated. PCR was performed following oligo(dT) reverse-transcription using the primers indicated in the schematic. Actin was amplified as a control. (D) HeLa cells were transfected with control or Ars2 siRNAs (Ars2-1, Ars2-2), RNA was isolated after 3 days and Northern blotting was performed for the HIST2H2BE transcript. Left - Total HIST2H2BE mRNA was detected with a 34 nucleotide probe designed to hybridize selectively to the HIST2H2BE 3′UTR upstream of the stem-loop. Right - To detect HIST2H2BE transcripts that were not properly cleaved, the membrane was stripped and re-probed with a probe generated by nick translation of a cDNA of the HIST2H2BE 3′UTR. (E) Additional Northern blots were performed on RNA isolated from HeLa cells transfected with Ars2 siRNAs (Ars2-1, Ars2-2) or control siRNA. Blots were probed with oligonucleotide probes within the open reading frames of histone H2B (left) or H2A (right) genes and therefore detect multiple conserved transcripts. (F) HeLa cells were transfected with control or three different Ars2 siRNAs (Ars2-1, Ars2-2, Ars2-3) and proteins were harvested after three days. Western blotting was performed for the four core histone proteins. Knockdown was confirmed by probing for Ars2 and Actin was used as a loading control. (G) Cell cycle analysis was performed on HeLa cells three days after transfection with control or three independent siRNAs targeting Ars2 (Ars2-1, Ars2-2, Ars2-3). Quantification of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle is shown in the upper right corner of each histogram. In agreement with these data the amount of DNA per cell remained unaltered following Ars2 knockdown (43.3 pg/cell in Ars2 siRNA vs. 42 pg/cell in control).

Figure 4. The Ars2 bound RNA 7SK promotes proliferation yet opposes proper replication-dependent histone mRNA 3′ end processing

(A) HeLa cells were crosslinked and immunoprecipitation was performed using control or Ars2 antibodies. RNA was isolated from the precipitate and Northern blotting was performed with a probe specific for 7SK RNA. tRNA^Tyr was probed to demonstrate the specificity of the Ars2-7SK interaction. Relative quantification of bands is displayed below each blot with input set to 1. 100 ng of total RNA was used for input. (B) HeLa cells were transfected with siRNAs targeting Ars2 (Ars2-2), 7SK, or a control siRNA. Two days later cells were collected, counted and re-seeded in triplicate into 6 well plates at equal densities. Cell counts were performed over the course of the next four days (day 0–4). Data points represent the average number of cells per well from two independent experiments +/− standard deviation. Inset - HeLa cells were transfected with siRNA targeting 7SK RNA or control siRNA and three days later RNA was isolated. Northern blotting was performed to confirm knockdown of 7SK. tRNA^Met was probed as a loading control. (C) HeLa cells were transfected with siRNA targeting 7SK or control siRNA and RNA was isolated three days later. cDNA synthesis was performed using either oligo(dT) or random hexamer primers and TaqMan®-based qPCR was used to determine changes in the levels of polyadenylated (poly(A)) and total HIST3H2A mRNA. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (D) Hela cells were transfected with siRNA targeting Ars2 (Ars2-2), 7SK or a control siRNA and RNA was isolated three days later. cDNA synthesis was performed using oligo(dT) primers and TaqMan®-based qPCR was used to determine changes in the levels of polyadenylated HIST2H2BE (H2BE) and HIST1H3H (H3H) mRNA. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (E) Northern blotting was used to determine the effect of 7SK depletion on total levels of histone H2B transcript.

Figure 5. Reciprocal interactions of 7SK RNA with Ars2 and CDK9

(A) Hela cells were transfected with siRNA targeting Ars2 (Ars2-2), CDK9 or a control siRNA and RNA was isolated three days later. cDNA synthesis was performed using oligo(dT) primers and TaqMan®-based qPCR was used to determine changes in the levels of polyadenylated HIST2H2BE (H2BE) HIST1H2AG (H2AG) and HIST1H3H (H3H) mRNA. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (B) HeLa cells were treated with 1μg/mL actinomycin D or DMSO control for one hour and then crosslinked. Immunoprecipitation with antibodies against Ars2, CDK9 or a control antibody followed by Northern blotting for 7SK was performed. U1 snRNA, which binds Ars2, was probed to insure Ars2 RNA binding was not compromised by actinomycin D treatment. Detection of 7SK in Ars2 immunoprecipitates increased 5.7 fold following actinomycin D treatment while detection of U1 snRNA only increased 1.4 fold, similar to the increase in non-specific U1 binding seen in control immunoprieipitates (1.3 fold increase). Detection of 7SK in CDK9 immunoprecipitates decreased 7.1 fold following actinomycin D treatment. A shorter exposure time was used to retain linearity of 7SK signal in CDK9 immunoprecipitates (right blot). tRNA^Tyr was probed to demonstrate the specificity of the Ars2-7SK and CDK9-7SK interactions. 100ng of total RNA was used as input. (C) IL-3-dependent Bax^−/−Bak^−/− cells were grown in the presence of IL-3 until day 0, at which point media was removed, cells were washed with PBS and resuspended in media without IL-3. Cells were maintained for 14 days in IL-3 deficient media, followed by restimulation with IL-3 at day 14.5. At the time points indicated aliquots were removed from the culture and RNA was extracted for Northern blotting with a probe for 7SK. The membrane was stripped and reprobed for U6 and U11 as loading controls. (D) IL-3-dependent Bax^−/−Bak^−/− cells were subjected to IL-3 withdrawal and restimulation as in (B). Aliquots of the culture were removed at the indicated time points and protein was extracted in RIPA buffer. An equal amount of total protein was run on SDS-PAGE gels and Western blotting was performed for Ars2 and CDK9. (E) HeLa cells were transfected with control, Ars2 or 7SK siRNAs. Two days later cells were washed with PBS and fresh media was added containing 10% serum or 0.1% serum. Twenty-four hours later RNA was extracted, reverse transcribed with oligo(dT) primers and TaqMan®-based qPCR was performed to determine relative levels of polyadenylated HIST2H2BE and HIST3H2A. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control. (F) Hela cells were co-transfected with siRNAs targeting Ars2 or CDK9 plus siRNAs targeting 7SK or control siRNA. Three days later RNA was extracted, reverse transcribed with oligo(dT) primers and TaqMan®-based qPCR was performed to determine relative levels of polyadenylated HIST2H2BE and HIST1H2AG. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control.