Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells.

Abstract

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 ...

Processing of Mammalian rRNA Precursors at the 3′ End of 18S rRNA

“…The experiments were carried out with mouse L929 fibroblast-like cells (ATCC, CCLl), cultured in Dulbecco's minimal essential medium, containing 5 % fetal calf serum and supplemented with antibiotics (penicillin and streptomycin) and glutamine. Transient transfection with the plasmids carrying the mouse rDNA minigene constructs was achieved by a DEAE-Dextran method, carried out in suspension as described previously [21]. Under standard conditions, DEAE-Dextran was used at 0.04% (final concentration) and the plasmid DNA at 8 pg/dish transfected cells.…”

“…Under standard conditions, DEAE-Dextran was used at 0.04% (final concentration) and the plasmid DNA at 8 pg/dish transfected cells. Routinely, the expression of the transfected rDNA minigenes was investigated 48 h after transfection, but similar levels of transcripts and their processing products were observed at 24-96 h. Taking advantage of the earlier observation that low concentrations of actinomycin D markedly stimulated the transcription and processing of transfected rRNA minigenes [21,291, transfected cells were routinely treated for 2 h with 0.05 pg/ml actinomycin D at 24 h before harvesting, to facilitate detection of the RNA product processed at the 3' end of 18s rRNA.…”

“…The structure of rRNA minigenes has been described in detail [21]. The basic mouse rRNA minigene, designated as pW (Fig.…”

“…The human rDNA insert (containing truncated 18s rRNA and ITS 1 sequences) from the plasmid construct pWH/S. Ild [21] (Fig. 2), in which the 21-nucleotide region of complementarity to U20 was replaced by a 6-nucleotide foreign sequence, was obtained with the oligonucleotide TGTTACGACTTTTACAGATCTGA-CCGTCTTCTCAGC (the foreign sequence is in bold characters).…”

“…We have recently developed an in vivo system for the analysis of pre-rRNA processing in mammalian cells using the transient expression of rRNA minigenes (with mouse promoter and terminator signals) transfected into cultured mouse cells [21]. These studies showed that truncated mouse or human rDNA transcripts are correctly processed at several mature rRNA termini.…”