PROCESSING OF TRUNCATED MOUSE OR HUMAN RIBOSOMAL-RNA TRANSCRIBED FROMRIBOSOMAL MINIGENES TRANSFECTED INTO MOUSE CELLS

The processing of pre-rRNA in eukaryotic cells involves a complex patt ern of nucleolytic reactions taking place in preribosomes with the par ticipation of several nonribosomal proteins and small nuclear RNAs. Th e 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 ex pression 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 thes e transcripts is analyzed by the use of reporter sequences flanking th e rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (I TS) 1,5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed pred ominantly to molecules coterminal with the natural mature rRNAs plus m inor products corresponding to cleavages within ITS 1 and ITS 2. To de lineate cis-acting signals in pre-rRNA processing, we studied series o f more truncated human-mouse minigenes. A faithful processing at the 1 8S rRNA/ITS 1 junction can be observed with transcripts containing onl y the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucl eotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleo tides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the t ranscript. 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 tru ncation of the 28S rRNA segment to 217 nucleotides abolishes processin g. Minigene transcripts containing most internal sequences of either I TS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not proces sed, suggesting that the cleavages in vivo within either ITS segment a re dependent on the presence in cis of mature rRNA sequences. These re sults show that the major cis signals for pre-rRNA processing at the 1 8S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited c ritical length of the respective mature rRNA and adjacent spacer seque nces.