Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Abstract

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.