Synthesis and processing of RNA by isolated human placental nuclei

Conditions for isolating intact and active nuclei from human term pacenta and for studying their transcription products are described. The isolated nuclei can synthesize cell-free RNA for a prolonged period at 29 degree C in a medium containing 100 mM KCl and 5 mM MgCl2. Actinomycin D inhibited 92 per cent of RNA synthesis, whereas approximately 60 per cent of RNA synthesis was sensitive to alpha-amanitin. When nuclei were incubated at 29 degrees C for 1 h, about 27 per cent of the newly synthesized RNA was released into the medium outside the nucleus. Analysis of this released material by affinity chromatography on an oligo(dT)-cellulose column revealed that 2.4 per cent of the total released RNA was adsorbed at high salt concentration. Most of this fraction was eluted with a low-salt buffer at 45 degrees C and the remainder by 50 per cent formamide, conditions that are necessary for elution of poly(A)-containing mRNP particles from oligo(dT)-cellulose. These results show that placental nuclei incubated in vitro synthesize poly(A)-containing RNA, which is released as a protein-bound complex. This procedure allows exploration of changes in mRNA release during placental development.