RNA synthesis and processing in isolated HeLa cell nuclei

Isolated HeLa cell nuclei have been characterised in terms of their ability to transcribe, process and transport RNA. In terms of transcription, it was found that all three RNA polymerases were active in the isolated nuclei. The size and nuclear location of the products of RNA polymerase I and III suggested that transcription by these polymerases was occurring normally in vitro. However, the RNA synthesised by RNA polymerase II was found to be much smaller than expected from the reported size of HeLa cell transcription units, when analysed in denaturing gradients. This was in contrast to the results of Sarma et al (1976) which showed that the size of RNA synthesised by RNA polymerase II in isolated HeLa cell nuclei is large when analysed under non-denaturing conditions. A number of possible reasons for this small size of RNA were examined. The results obtained indicate that this small size of RNA polymerase II product was probably not due to;- i. A slow elongation rate by RNA polymerase II resulting in a "nascent transcript profile." ii. Degradation of RNA in the isolated nuclei. iii. The absence of nuclear and cytoplasmic factors during incubation of the nuclei. iv. Degradation of the DM template during isolation and incubation of nuclei. It was found, however, that the state of the chromatin template was important in determining the size of RM transcribed. Thus, addition of acetyl CoA to isolated nuclei, which acetylated the histones, caused an increase in the size of RNA polymerase II product. On the other hand methylation of histones with Ado-Met vitro was correlated with a decrease in the size of RM. It was also found that the ionic content of the incubation medium affected the size of RNA transcript synthesised by RNA polymerase II. In particular, substituting 90 mM (NH4)2SO4 for 75 mM KCl in the incubation medium increased the size of RNA. This effect is discussed in terms of recent results which suggest that the small size of RNA polymerase II transcript commonly observed in vitro might be due to premature termination of transcription. The small RNA transcribed by RNA polymerase II in vitro appears to be stable. However, hnRNA prelabelled in vivo reduced in size during incubation of isolated nuclei. Some of this RNA is released from the nuclei during incubation. This release of RNA was examined to determine whether it represented the specific transport of mRNA. Although the size of released RNA particles, their ability, and the size of released RNA were consistent with mRNA transport, other features were more consistent with the leakage of hnRNP particles. The released RNA resembled hnRNA in terms of binding to poly(U) Sepharose, and the protein associated with the released RNA were similar to hnRHP particle proteins. Although the released RNA had messenger activitiy in a wheat germ cell free translation system, it is not possible to rule out mRNA contamination as the cause of this stimulation. It therefore appears that the isolated nuclei system of Sarma et al (1976) may not be ideal either for examining the transcription or the processing and transport of mRNA. On the other hand, the results obtained in the present study suggest ways in which this system might be modified in order to achieve full length transcription by RNA polymerase II in vitro, and study the processing and transport of these transcripts