Synthesis, methylation, and capping of nuclear RNA by a subcellular system

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Abstract
A subcellular system [mouse L cells] is described which is capable of in vitro synthesis of large nuclear RNA and the formation of both cap I [m7G(5'')pppXmpYp] and cap II [m7G(5'')-pppXmpYmpZp] structures. This system, which consists of partially purified intact nuclei and residual cytoplasmic tags, carries out both guanosine addition, utilizing GTP, and the appropriate methylation reactions, utilizing S-adenosylmethionine as the methyl donor. The general structure of the caps was verified by analyses of methylated derivatives recovered after RNase T2 hydrolysis and after digestion with P1 nuclease, bacterial alkaline phosphatase, and nucleotide pyrophosphatase. Cap formation in large nuclear RNA species was closely associated with transcription, as indicated by .alpha.-amanitin sensitivity and a requirement for the presence of all 4 nucleoside triphosphates. Recovery of a class of cap II structures, in which only the methyl group at position Y is labeled, as well as cap II structures in which all methylated constituents are labeled, indicates the presence of at least 2 independent methylation events in the vitro system.