Alternate Ribosomal-Rna Processing Pathways in Hela Cells.

Abstract

Restriction fragments of the human genes for ribosomal RNA (rRNA), cloned in pBR322, were used to correlate the gene map with the rRNA primary transcript, processing intermediates and mature rRNA's. Southern blots of cloned fragments representing 18S sequences and external transcribed spacer sequences were hybridized to labeled HeLa cell total RNA and cytoplasmic RNA in order to establish the boundaries of the transcribed area and of the mature 18S coding sequences. Electrophoretically separated HeLa RNA was blotted onto diazo-paper and hybridized to subcloned restriction fragments to reveal processing intermediates. All known intermediates were detected. In addition, an abundant new 7.0 kb intermediate was detected by hybridization to subclones containing only external transcribed spacer sequences. A subclone which contains only 18S coding sequences was constructed and used to establish the presence of 18S sequences in the novel 7.0 kb RNA. Blots of HeLa cytoplasmic and nuclear RNA revealed that the 7.0 kb RNA, like other known rRNA precursors, is present in the nucleus and absent from the cytoplasm. Hybridization of the RNA blots to cloned DNA containing internal transcribed spacer sequences revealed the 5.8S ribosomal RNA and an abundant 0.7 kb RNA. It is proposed that this 0.7 kb RNA represents a processing intermediate in the pathway to 5.8S rRNA. Cross-hybridization of the cloned sequences to previously described rRNA precursors could explain these data. Therefore, Southern blot experiments were performed to detect cross-hybridization between the different portions of the rRNA transcriptional unit. Areas of cross-homology were detected at the 3' end of the 28S coding sequences, but this cannot account for the observed new RNA species. No other areas of cross-hybridization could be detected. A new model for ribosomal RNA processing in HeLa cells is proposed to account for these RNA species. This model allows for the initial cleavage of the primary transcript to occur either at the 5' side of the 18S sequences, as in the currently accepted scheme, or at a site within the internal transcribed spacer.