The human cytoplasmic RNA terminal U-transferase ZCCHC11 targets histone mRNAs for degradation

Abstract

Inhibition of eukaryotic DNA replication leads to the rapid suppression of histone synthesis, via 3' uridylation of cytoplasmic histone mRNAs followed by their Lsm1-7-mediated decapping and degradation. Here we show that the human cytoplasmic RNA terminal U-transferase ZCCHC11, recently implicated in microRNA metabolism, associates with replication-dependent histone mRNAs. Knockdown of ZCCHC11 selectively blocked histone mRNA degradation following inhibition of DNA replication, whereas knockdown of PAPD1 or PAPD5, previously proposed as candidate histone mRNA U-transferases, had no such effect. Furthermore, a reduction in the proportion of histone transcripts that were uridylated was observed following ZCCHC11 knockdown. Our data indicate that ZCCHC11 is the terminal U-transferase responsible for targeting human histone mRNAs for degradation following inhibition or completion of DNA replication.

FIGURE 1.

ZCCHC11 is a cytoplasmic protein and specifically associates with replication-dependent histone mRNAs. (A) Cytoplasmic (cyto) and nuclear HEK293T cell extracts were analyzed by Western blotting with a ZCCHC11-specific antibody. α-Tubulin and topoisomerase IIα served as controls for cytoplasmic and nuclear localization, respectively. (B) ZCCHC11 was immunoprecipitated from cytoplasmic extracts in the presence (+) or absence (−) of polyclonal anti-ZCCHC11 antibody. (C) Following formaldehyde cross-linking RNPs were immunoprecipitated (IP) using specific antibody or no antibody (ab). Precipitates were subjected to RT-PCR using primers specific for the ORF of replication-dependent histone HIST2H3 (H3). Products were separated by agarose gel electrophoresis. M, molecular weight marker. (D) As in C, but H3 mRNA (and GAPDH mRNA as a control) were analyzed using real time RT-PCR. Data (mean of three biological replicates, ±SD) are expressed relative to the signal obtained in the absence of specific antibody.

FIGURE 2.

ZCCHC11 is required for efficient degradation of replication-dependent histone mRNAs upon inhibition of DNA replication. (A,B) ZCCHC11 was knocked down using shRNAs directed against its 3′ UTR. A scrambled shRNA control (ctrl) was used in parallel. Forty-eight hours following shRNA transfection, HEK293T cells were transfected with either empty (e), WT, or catalytically inactive mutant (mut) ZCCHC11 expression vector as indicated. The expression of ZCCHC11 was analyzed by Western blot using anti-ZCCHC11 (and anti-tubulin as a control; A) and by quantitative RT-PCR (B) 72 h after shRNA transfection. (C) As in B, but cells were treated with (+) or without (−) 5 mM hydroxyurea (HU) for 30 min before being harvested. Total RNA was isolated and analyzed by real time RT-PCR. H3 mRNA values (mean of five biological replicates ±SD) were normalized to GAPDH mRNA and expressed relative to the RNA amount in untreated samples, which was arbitrarily set to 1.0. (D) ZCCHC11 levels (and α-tubulin as a loading control) were monitored by Western blotting extracts of cells treated (+) or untreated (−) with 5 mM HU for 30 min.

FIGURE 3.

The impact of ZCCHC11 knockdown on histone mRNA uridylation. (A) Overview of the cRACE procedure used to capture capped HIST2H2AC transcripts (black) and degradation intermediates (gray). Arrows indicate the position of the PCR primers used. (B) cRACE was performed on TAP-treated RNA isolated from HeLa cells harvested at the end of S phase following shRNA plasmid transfection. The percentage of cRACE products containing terminal uridyl residues is shown for control and ZCCHC11 knockdown cells (n = 14/46, 8/48, respectively). (C) Diagram indicating the positions of uridyl residues detected by cRACE.

FIGURE 4.

Effect of PAPD1 and PAPD5 knockdown on the degradation of replication-dependent histone mRNAs upon S phase arrest. (A) HEK293T cells were transfected with control (ctrl) or specific shRNAs directed against PAPD1, PAPD5, or Lsm1. Seventy-two hours after transfection, cells were treated with (+) or without (−) 5 mM hydroxyurea (HU) for 30 min. RNA was analyzed as in Figure 2C. The mean of at least three biological replicates ±SD is indicated. (B) Steady-state H3 mRNA levels in asynchronous cells transfected with the shRNA expression vectors indicated were compared to those in cells expressing the scrambled control shRNA (ctrl).

FIGURE 5.

ZCCHC11 expression negatively correlates with H3 expression during S phase. HeLa cells were blocked in early S phase by a double thymidine block and then released. (A) The cell cycle distribution of propidium iodide stained cells was analyzed by flow cytometry at the indicated times after release from the block, and in asynchronous cells. (B) Upon removal of the block, total RNA was isolated at time points indicated and analyzed by real time RT-PCR using specific oligonucleotides. H3 and ZCCHC11 (upper panel) and H3, PAPD1, and PAPD5 (middle panel) mRNA levels were normalized to GAPDH mRNA and expressed in relation to RNA obtained from cells at the time when the block was removed (0 h). ZCCHC11 and α-tubulin protein levels were measured by Western blotting at the same time points (lower panel).