In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells

Abstract

Isolated rat liver nuclei were incubated under appropriate conditions in the presence of 0.5 μg/ml α-amanitin and an RNAase inhibitor prepared from cytosol fraction, together with α- 32P-UTP or α- 32P-CTP and three other nucleoside triphosphates. RNA extracted by an SDS-hot phenol procedure was fractionated with sucrose density gradient centrifugation followed by acrylamide gel electrophoresis. Fingerprint analysis of the in vitro synthesized “5S” RNA, which was slightly larger than mature 5S RNA on gel electrophoresis, showed that it contained all the sequences of mature 5S RNA except for the oligonucleotide at the 3′ end. Instead, it contained two additional spots which were not present in mature 5S RNA. Analysis of the extra spots revealed that they were derived from the 3′ end of the in vitro synthesized “5S” RNA, which was sequenced tentatively as -CUU GAUGCUUUoh (extra sequence underlined). The 5′ end of the product was (p)pGU—. Isolated HeLa cell nuclei synthesized similar sized “5S” RNA under the same conditions. We conclude from these results that in isolated nuclei of these mammalian cells, RNA polymerase III starts transcription of 5S RNA gene at the same site as the 5′ end of mature 5S RNA, proceeds toward the 3′ direction and stops at a site probably 8 nucleotides downstream from the 3′ end of mature 5S RNA. Experiments with a short pulse and with various “chases” have demonstrated the presence of a short-lived precursor 5S RNA which is similar in size and sequence to in vitro “5S” RNA, suggesting that 5S RNA is synthesized in vivo as a longer precursor molecule as demonstrated in this in vitro system, and is rapidly processed into mature 5S RNA.