RNA synthesis and processing reactions in a subcellular system from mouse L cells.

Abstract

A subcellular system from mouse L-cells has been used to study RNA synthesis and processing in vitro. The nuclei in this system incorporate nucleoside triphosphates into RNA with high yield for more than 120 min. The capacity for RNA synthesis is stable for extended periods at 4 degrees C. All three RNA polymerases contribute to the overall synthetic activity as shown by differential inhibition with alpha-amanitin. The in vitro labeled RNA contains about 15% of polyadenylated RNA. The non-polyadenylated RNA shows molecules in the range of larger than 20 S down to 4-5 S. The polyadenylated RNA exhibits mainly transcripts around 18 S and below 8 S. Methylation of nucleoside bases and the ribose 2'-OH group including 5'-caps is performed in vitro as well. Base methylation and 5'-cap methylation are partially sensitive to alpha-amanitin. 28% of the methyl groups are found in polyadenylated RNA being distributed throughout molecules of all sizes. The methylated non-polyadenylated RNA shows peaks at 45 S, around 28 S and 18 S, and a very prominent low-molecular weight RNA peak. Addition of the poly(A) tract to RNA molecules in vitro is revealed by the presence of [3H]-uridine-labeled polyadenylated RNA. The poly(A) tract was isolated and analyzed on polyacrylamide gels. Its maximum length coincides with an in vivo poly(A) marker indicating the addition of about 150-200 nucleotides. Poly(A) addition is possible on pre-existing RNA chains, preferably on 3'-oligo(A) tracts. This process is insensitive to alpha-amanitin. In addition, the specificity of polyadenylation may be relaxed since incorporation of [3H]UTP into polyadenylated RNA is only reduced to about 50% under conditions (1 microgram alpha-amanitin/ml) where RNA polymerase II is inhibited. A small fraction of the in vitro labeled RNA binds to polysomes which can be recovered from the cytoplasm adhering to the nuclei. This RNA contains poly(A) and is enriched in base methylations and 5' end caps. It can be dissociated from the polysomes by EDTA. It is likely to be in vitro labeled and maturated mRNA.