The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.

rRNA processing pathways in humans have been reinvestigated through systematic Northernblot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenine-labeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41 S, 32s and 21 S pre-rRNA, two major pre-rRNA fractions were identified; a '30s' (about 5800 nucleotides) precursor to 18 S rRNA containing an external transcribed spacer at the 5' end (ETS) and internal transcribed spacer (ITS) 1 sequences and a '12s' (about 950 nucleotides) precursor to 5.8s rRNA containing ITS 2 sequences. These pre-rRNA species do not react with probes located near the 3'-terminal segments of ITS 1 or ITS 2, thus suggesting that processive endonuclease cuts occur within ITS spacer sequences. The simultaneous occurrence of at least two alternative 45s prerRNA processing pathways is deduced, which correspond to a different temporal order of endonuclease attack at the sites located near the 5' end of 18s rRNA and within ITS 1. In-vivo labeling experiments with [Wluridine revealed that inhibition of protein synthesis with cycloheximide abolishes the endonuclease cut at the 5' end of 18 S rRNA and the formation of 41 S pre-rRNA, while the cut within ITS 1 and the processing to 32s and '30s' pre-rRNA remains relatively unaltered.Three out of the four major rRNA species in eukaryotes are generated from a single pre-rRNA transcript processed to yield the sequences of the mature S rRNA, 5.8s rRNA and L-rRNA [l, 21. The remarkably conserved pattern in the arrangement of rRNA segments within the primary transcript,L-rRNA -3'-ETS (ETS, external transcribed spacer; ITS, internal transcribed spacer) initially suggested the vectorial operation of subsequent endonucleolytic pre-rRNA processing steps, each of them triggering the next one and resulting in a single processing pathway [l]. Indeed, the requirement for an initial endonucleolytic removal of a minor part of the 5'-ETS and virtually the complete removal of 3'-ETS from the primary pre-rRNA transcript was documented for mammalian cells , although it could not be observed in Xenopus [8,9]. However, it was soon realized that the further processing of pre-rRNA could involve a varying order of endonuclease attacks, thus resulting in the simultaneous operation of alternative processing pathways 11, lo]. The role of the observed alternative pre-rRNA processing pathways in the growth and/or function of eukaryotic cells remains to be elucidated.Moreover, the realization that the removal of ITS 1 and ITS 2 from pre-rRNA does not merely result from cleavages at the termini of mature rRNA, but could also involve endonucleolytic cuts within each internal spacer, added further complexity to the problem. Thus, the presence of pre-rRNA species containing truncated internal spacer sequences has been documented for yeasts [ll, 121, mouse [13, 141 and rat [15 -171 cells and tissues.Although it is presumed that pre-rRNA processing in human cells is identical to that in other mammals, direct ...

The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined. The NTS sequences of both species were found to be 93% homologous. The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs. Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1). The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs. The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start. In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS. The strands of the yeast NTS reveal a remarkable bias of G and C-residues. Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W. and Wu, R (1979), Plasmid 2, 536-554). This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2.

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.Although the transcription of rRNA genes in eukaryotes has been elucidated to considerable detail (37,42,43), the molecular mechanisms of pre-rRNA processing remain largely unknown (44). The primary transcript of rRNA genes undergoes in vivo a complex pattern of successive nucleolytic cleavages resulting in the elimination of the transcribed spacer sequences and the formation of the mature rRNAs (18). While this process takes place within preribosomes and involves the participation of several nonribosomal nucleolar proteins and small nucleolar RNAs (reviewed in references 9, 10, 18, 44, 48, and 49), it seems likely that the specificity of the cleavages is largely determined by definite structural features of pre-rRNA molecules. This possibility has been stressed by studies on the initial endonuclease cleavage ...