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.