Characterization of pre-rRNA components in ribosomal precursor particles from macronuclei of Tetrahymena thermophila.

Ribosomal precursor particles were extracted from purified macronuclei of Tetrahymena thermophila and separated in sucrose gradients. The RNA components of major particle fractions were isolated and analyzed by Northern blot hybridization using cloned rDNA fragments. A tentative scheme of preribosome maturation was established based on the RNA constituents and corresponding processing steps occurring in the different particle classes. The primary transcript of ribosomal genes, the unspliced precursor rRNA, was found in some experiments in the upper region (less than 40S) of sucrose gradients run for short times. This is in accordance with earlier results by others indicating that slowly sedimenting ribonucleoprotein (RNP) structures may exist as a transitory stage of preribosome formation. Usually, however, unspliced pre-rRNA was only found in 80S preribosomes, where splicing occurred as indicated by the presence of splice intermediates and products only in this fraction. In addition, the further processing of spliced pre-rRNA at three major sites in variable temporal order took place in the 80S preribosomes, i.e., (i) the cleavage at or near the 5'end of the 17S rRNA sequence, (ii) the central cleavage in the internal transcribed spacer (ITS2) between the 5.8S and 26S rRNA sequence, and (iii) the cleavage in the ITS1 at or near the 3' end of the 17S rRNA sequence. Only the latter event was found to result more or less immediately in the division of the 80S preribosomes into separate precursors (p40S and 60S) of the small and large ribosomal subunits. If the alternative pre-rRNA cleavage site in the ITS2 was used first the 80S preribosomes retained their integrity. The conversion of the p40S precursors into nuclear 40S subribosomal particles was correlated with the processing of pre-17S rRNA into 17S rRNA. In the 60S ribosomal precursor particles the processing of pre-26S rRNA, including the formation of precursors (ITS and 7S RNA) to 5.8S rRNA, occurred. A substantial proportion of 26S rRNA molecules isolated from these particles already contained the central hidden break as indicated by the presence of 26S rRNA alpha- and beta-subfragments. The major pre-rRNA processing by-products, IVS and ETS RNA, were partly associated with preribosomes and partly present as free RNAs in the supernatant of sucrose gradients. This indicates that they are liberated and degraded mainly outside the particles in which they are formed. In contrast, the initiation fragment (IF), a small promoter-proximal transcript, was exclusively associated with large particles.(ABSTRACT TRUNCATED AT 400 WORDS)