Association of RNA with the cytoskeleton and the nuclear matrix

Abstract

Heteronuclear RNA (hnRNA) is preferentially associated (76%) with the nuclear matrix in mammalian cells. Active mRNA, in the form of polyribosomes, is associated (greater than 97%) with the cytoskeletal framework. In this report, we present evidence that the association of both hnRNA and mRNA with structural networks of the cell may be essential features of gene expression. To study the association of polyribosomes with the cytoskeletal framework, cytochalasin D was used to release mRNA from the cytoskeletal framework. Protein synthesis was inhibited by cytochalasin D in direct proportion to the release of mRNA. The released mRNA is unaltered in its translatability as measured in vitro but is no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurs on polyribosomes that are reduced in amount but display a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary, though not sufficient, for translation. Further fractionation of the cytoskeletal framework separates nuclear constituents into three distinct protein fractions. Chromatin proteins and 94% of the DNA are released by 0.25 M-ammonium sulphate after inter-nucleosomal DNA is cut with DNase I. The resulting structure retains 76% of the hnRNA in the form of ribonucleoprotein and is designated the RNP-containing nuclear matrix. The proteins of hnRNP complex are those associated with the nucleus only if RNA is intact. These proteins and 97% of the hnRNA are released after brief digestion with RNase A. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in the organization of the nuclear matrix. Electron micrographs of resinless sections show the interior of the matrix to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are densely covered with 20-30 nm electron-dense particles, which may contain the hnRNA. The RNP-depleted matrix is disordered and the interior fibres aggregated. These results suggest that hnRNA is involved in the spatial organization of the interior of the nuclear matrix.