Characterization of mouse 45S ribosomal RNA subspecies suggests that the first processing cleavage occurs 600 +/- 100 nucleotides from the 5' end and the second 500 +/- 100 nucleotides from the 3' end of a 13.9 kb precursor

Abstract

Mouse fibroblasts labeled 1-9 h with 3H-uridine contained radioactive 45S rRNA subspecies of 13.9, 13.3, and 12.8 kb, as determined by hybrid-selection with rDNA plasmids and by electrophoresis in agarose-formaldehyde. The 13.9 kb subspecies contained 5' and 3' terminal rDNA sequences known from the work of Grummt and colleagues to be at or near the ends of the primary transcript. The 13.3 kb subspecies contained the 3' terminal sequence but lacked the 5' terminal sequence. The 12.8 kb subspecies lacked both terminal sequences. Washed nuclei produced one discrete species of 13.9 kb. The results suggested that synthesis of the primary transcript terminated 500 +/- 100 nucleotides beyond the 3' end of 28S rRNA, that the first processing cleavage occurred 600 +/- 100 nucleotides from the origin of synthesis, and the second cleavage occurred near the 3' end of 28S rRNA. Changes in relative radioactivities among the subspecies after serum stimulation or after treatment with low concentrations of cycloheximide suggesting that processing was not perfectly coupled with synthesis and that cycloheximide inhibited one cleavage more than others.