RNA species present on rat liver nuclear matrices were investigated. Nuclear matrices prepared by extensive digestion of isolated nucleii with DNase and RNaseA followed by low and high (2M NaC1) salt washes were labelled in vitro with T4 RNA ligase and [5'-32P]pCp and the labelled RNA analysed by gel electrophoresis. Despite the extensive RNaseA treatment, a prominent RNA species migrating as a heterodisperse band of 220-300 nucleotides (termed MX220-300), was observed--only minor amounts of other RNA molecules were seen. A comparison of RNA isolated from in-vitro labelled nuclear matrices with isolated matrix RNA that was subsequently labelled, indicated that part of MX220-300 was preferentially exposed on the nuclear matrix structure. Analysis of MX220-300 indicated that it was composed of a polyadenylic acid moiety hydrogen bonded to a smaller molecule of polyuridylic acid. No evidence was found for the presence of guanosine or cytosine residues. Control experiments in which labelled polyuridylic acid was added to nucleii prior to the preparation of MX220-300, virtually excluded the possibility that the partial double stranded RNA structure was an artefact of matrix preparation. An analysis of proteins in the nuclear matrix structure that interact with double stranded (ds)RNA showed at least 2 proteins having molecular weights of 62K and 66K daltons that recognized and bound polyadenylic/polyuridylic acid. Competition experiments with unlabelled polyinosinic/polycytidylic acid indicated that these proteins specifically recognized the dsRNA structure. The 62K and 66K dalton matrix proteins that specifically bound dsRNA were observed in nuclear matrices prepared from HeLa, Ehrlich ascites tumor and rat liver cells. It is not known whether these matrix located dsRNA binding proteins have 2-5A synthetase activity. The relevance of the above findings to the 2-5A system will be discussed.
|Number of pages
|Progress in Clinical and Biological Research
|Published - 1985