Mercurated nucleotides: essessment of a new tool to study RNA synthesis and processing in isolated nuclei

• Published in: Nucleic acids research 1977-12, Vol.4 (12), p.4465-4480
• Main Author: Schäfer, Klaus P.
• Format: Article
• Language: English
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/4.12.4465

Mercurated pyrimidine nucleotides have been used to study RNA synthesis and processing in isolated nuclei from mouse L cells. 5-mercuridine triphosphate (5-Hg-UTP) or 5-Hg-CTP are accepted as substrates by the purified RNA polymerases (I+III) and (II) from mouse cells, respectively, as well as by the enzymes still bound to the nuclear chromatin. In nuclei, RNA synthesis in the presence of Hg-UTP is reduced 60–70% of a control. 30–60% of RNA labeled in vitro with (3H)UTP in isolated nuclei is not retained on sulfhydryl sepharose columns. Sucrose gradient analysis reveals a size distribution of the non-bound RNA similar to non-mercurated control RNA. Hg-RNA is found in a single peak from 47–10S. Chase experiments indicate that this RNA is the original transcript. It is argued that Hg-nucleotides may cause premature chain termintion. Nethylation of RNA in vitro by S-adenosyl methionine ((3H)SAM) is reduced to 75% of controls in the presence of Hg-UTP. Only 6% of the methyl groups appear in Hg-RNA. Polyadenylation is reduced as well. 15% of poly(A)(+)RNA are found in control assays whereas only 1% of Hg-RNA carries a poly(A) end added in vitro. These results limit the use of mercurated nucleotides for studies of nuclear RNA synthesis and processing.