FIG. 7.

SLBP is the only essential histone pre-mRNA processing factor missing from G₁ and G₂ nuclear extracts. (A) HeLa cells were synchronized by a double-thymidine block, and equal cell equivalents of nuclear, polyribosomal, and cytosol fractions were prepared from S phase (3 h after release [lanes 2, 5, and 8]), G₂ phase (8.5 h after release [lanes 3, 6, and 9]) and G₁ phase (14 h after release [lanes 1, 4, and 7]) and analyzed for SLBP by Western blotting. (B) The nuclear extracts prepared from S-, G₂-, and G₁-phase cells were analyzed for processing activity using the H2a-614 pre-mRNA as a substrate. Recombinant SLBP was added to the extracts in lanes 2, 4, and 6 prior to addition of the pre-mRNA substrate. RNA was prepared from the reaction mixtures, and the pre-mRNA (Unproc) and processed mRNA (Proc) were resolved by gel electrophoresis. The results are representative of three independent preparations of synchronous cells. (C) Flow cytometric analysis of the cells used for preparation of the S-phase (3 h), G₂-phase (8.5 h), and G₁-phase (14 h) extracts, respectively. Chromosome content is shown on the x axis, and cell number is shown on the y axis.