ISOLATION AND CHARACTERIZATION OF AN RNA-CONTAINING NUCLEAR MATRIX FROM TETRAHYMENA MACRONUCLEI

We have developed a procedure allowing the isolation of an RNA-containing nuclear matrix (RNM) from Tetrahymena macronuclei in high yield (~60%) by low- and high-salt extractions including DNase treatment. Electron microscopy reveals that the RNM still shows the nuclear sphere shape and is composed of condensed fibrillar-granular material in its interior, whereas the “residual nucleoli” located at the periphery and structurally linked to the “residual nuclear envelope” essentially consist of more loosened fibrillar material, including tiny fibrils (~4 nm in diameter). The protein pattern of the RNM, as revealed by electrophoresis on 10-15% NaDodSO4-polyacrylamide gradient gels, is nearly identical with that of the RNA-free nuclear matrix showing bands in the molecular weight region between 12000 and 300000 with one prominent band of 18 000 molecular weight. The majority of the RNA (~85%) included in the RNM consists of specific pre-rRNA fractions (A-D). Fraction A containing the primary stable transcription product of the rDNA seems to be almost completely retained in the RNM, whereas fractions B and D, the direct nuclear precursors to the cytoplasmic 26S and 17S rRNAs, are partly solubilized during RNM preparation. Moreover, some 5S rRNA and at least two specific low molecular weight (LMW) RNAs with low turnover (TA and Tc) are part of the RNM. One LMW RNA component (TD) which is contained in whole macronuclei is lost during RNM isolation, together with some material from the 5S region. [3H]Uridine-labeling kinetics of the RNA recovered in the RNM compared with those of the solubilized RNA show that the rapidly labeled RNAs are at first tightly bound to the nuclear matrix and then successively become more susceptible to solubilization during the extraction procedure. Our results are compatible with the view that processing and intranuclear translocation, at least of rRNA, occur along the nuclear matrix elements in a highly ordered process. © 1979, American Chemical Society. All rights reserved.