Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells

Highly purified steady state heterogeneous nuclear RNA from HeLa cells has been prepared by a new procedure. Detergent-washed nuclei are disrupted in 0.4 M ammonium sulfate, which also disociated contamination polysomes. The hnRNA remains bound to chromatin, which can be pelleted by gentle centrifugation. Ribonuclease inhibitors permit the preparation of very high molecular weight nuclear RNA. The hnRNA was cleaved with alkali. The poly(A)-containing fragments were separated from those containing oligo(A), and a cDNA copy was prepared. Hybridization of this nuclear cDNA to cytoplasmic mRNA showed that the scarce (complex) message sequences make up a larger proportion of nuclear RNA than of cytoplasmic RNA. In addition, at least 30% of the poly(A)-adjacent sequences in nuclear RNA have no apparent counterparts in the cytoplasm. cDNA prepared from hnRNA sedimenting faster than 45S under denaturing conditions gives similar results, showing the presence of both message and non-message sequences in very large transcripts. cDNA complementary to mRNA was separated into the abundant and scarce sequences, and hybridized separately to the poly(A)-adjacent sequences in nuclear RNA. The hybridization of the abundant sequence cDNA was used to set an upper limit on possible cytoplasmic contamination. Hybridization of the scarce cytoplasmic sequences are represented in nuclear RNA approximately once per cell.