ALTERNATIVE PRE-RIBOSOMAL-RNA PROCESSING PATHWAYS IN HUMAN-CELLS AND THEIR ALTERATION BY CYCLOHEXIMIDE INHIBITION OF PROTEIN-SYNTHESIS

rRNA processing pathways in humans have been reinvestigated through systematic Northern-blot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenine-labeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41 S, 32 S and 21 S pre-rRNA, two major pre-rRNA fractions were identified; a '30 S' (about 5800 nucleotides) precursor to 18S rRNA containing an external transcribed spacer at the 5' end (ETS) and internal transcribed spacer (ITS) 1 sequences and a '12S' (about 950 nucleotides) precursor to 5.8S rRNA containing ITS 2 sequences. These pre-rRNA species do not react with probes located near the 3'-terminal segments of ITS 1 or ITS 2, thus suggesting that processive endonuclease cuts occur within ITS spacer sequences. The simultaneous occurrence of at least two alternative 45 S pre-rRNA processing pathways is deduced, which correspond to a different temporal order of endonuclease attack at the sites located near the 5' end of 18S rRNA and within ITS 1. In-vivo labeling experiments with [C-14]uridine revealed that inhibition of protein synthesis with cycloheximide abolishes the endonuclease cut at the 5' end of 18 S rRNA and the formation of 41 S pre-rRNA, while the cut within ITS 1 and the processing to 32S and '30S' pre-rRNA remains relatively unaltered.