Transcription and processing of RNA from mouse ribosomal DNA transfected into hamster cells.

Abstract

Transcription of mouse genes coding for rRNA in CHO cells was promoter dependent at levels 3 to 10% of that of endogenous rRNA synthesis. Northern (RNA) and Si nuclease mapping analyses demonstrated that transcription proceeds through the entire gene segment coding for rRNA in transfected constructs and continues, at least in some cases, into the adjoining plasmid sequences. Si nuclease mapping also detected some processing cleavages in the transcripts, including those at the 3' terminus of 18S rRNA, those at the rapidly cleaved site at +650 in the external transcribed spacer, and those at a previously uncharacterized, rapidly cleaved site in the internal transcribed spacer. Deletion of sequences upstream or downstream from the promoter generally had no measurable effect on the level of transcription, but deletion of a 300-base-pair XhoI-XhoI fragment starting 1,287 base pairs from the transcription start site sharply increased the steady-state level of rRNA. Effects on processing were harder to test, because many intermediates are too unstable to detect even by Si nuclease mapping; however, the data suggest that RNAs with deletions in the external transcribed spacer are processed poorly at distal sites. Processing at some sites may thus depend on interactions involving distant segments of rRNA.The processing of mRNA precursors has been extensively analyzed in extracts and in cells (16,18). In contrast, comparable information on the processing of rRNA remains sparse. While it is known that rRNA processing in mammalian cells proceeds through a set of simple cleavages, no in vitro system which produces fully processed rRNAs has yet been devised. Even in vivo, the expression of transfected constructs of genes coding for rRNA (rDNA) is difficult to study because of the very high background of endogenous rRNA formation. One way to overcome this problem is to analyze the expression of transfected heterologous rDNA with probes unique to spacer sequences in the transfected rDNA (9). Even though rDNA transcription has been shown to be restricted by species-specific barriers (10, 14), it can be studied in cells or extracts of some closely related species (human and monkey [12], mouse and rat [20], and mouse and hamster [7]). In those cases, the spacer sequences are different enough to permit adequate discrimination of transcripts from endogenous and transfected rDNA. Recombinant rDNA plasmids. Figure 1 shows a schematic map of the subclones and deletion constructs used in the transient expression experiments. All plasmids used for transfection were cloned in the vector pUC18. Mouse clone numbers are as described elsewhere (2, 3). pCL3 was derived by subcloning the 3.2-kilobase (kb) SalI-SalI fragment of pMr974 (20) insert into the EcoRI site of clone 6 in the appropriate orientation. The resulting construct, containing the mouse rDNA promoter, 8 kb of upstream DNA, and an intact transcription unit through the first one-third of internal transcribed spacer 2 (ITS2), was the parent molecule for a number of constructs. pRD18...

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

“…Selective cleavage at three specific sites (including one at the 18S rRNA/ITS 1 junction) was reported also for an in vitro transcript of mouse rDNA (containing only 18S and ITS 1 sequences) digested with a purified nucleolar endoribonuclease (40). However, it should be noted that in our system, we do not observe the additional cleavages within 18S rRNA or at 55 nt within ITS 1 reported in earlier studies (36,40). Further, our results permit us to localize the minimal sequence requirements for faithful cleavage at the 18S rRNA/ ITS 1 junction.…”

“…Thus, cleavage in rat cells at or near the 5 [3'IGS] carrying the transcription termination signal [15]) and (ii) CAT sequences from pSV2-CAT (13), split by a synthetic polylinker (pl) transcripts, which lacked both the 3'-terminal region of 18S rRNA and all downstream sequences (46). In another study using mouse rDNA transfected into hamster cells, cleavage of the transcript near the 18S rRNA/internal transcribed spacer (ITS) 1 boundary was observed, together with an additional cleavage at about 55 nucleotides (nt) within ITS 1 (36). While these observations demonstrated that processing at either end of 18S rRNA can proceed in the absence of the other end, further dissection of the processing signals in cis was not carried out.…”

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

“…Thus, transfection of mouse rDNA into rat cells has shown that processing at the 5' end of 18s rRNA does not require most of the downstream 18s rRNA sequences 1131. Also, transfection of mouse rDNA into hamster cells revealed that processing at the 18s rRNA/internal transcribed spacer 1 (ITS 1) junction can take place without a complete 18s rRNA sequence [14]. In mammalian cells, the latter reaction occurs on a nucleolar 21s prerRNA, representing the last step in the production of mature 18s rRNA [15 -18).…”

In Vitro Processing at the 3′-Terminal Region of Pre-18S rRNA by a Nucleolar Endoribonuclease

“…These examples suggest that trimming processes may also be involved in the formation of mature rRNA products. Early cleavage events were also inferred from results obtained by using transfected mouse rDNA segments in CHO cells (19). In these studies, Si protection analysis detected processing cleavages which included the +650 site, the 3' end of 18S rRNA, and a rapidly cleaved site in the first internal transcribed spacer (ITS1).…”

“…The hybridization reaction mixtures (30 ,ul) contained the RNA transcript (0.15 ,ug), approximately 0.02 pmol of complementary end-labeled DNA, 100 ,ug of Escherichia coli tRNA, 80% deionized formamide, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 6.4), 0.4 M NaCl, and 1.0 mM EDTA. The hybridization reactions were denatured at 85°C for 15 min, totally immersed in a water bath, and incubated at 60°C for 3 h. A modification of the procedure of Berk and Sharp (2) (19) used Si nuclease protection analysis of rRNA cleavage products from a mouse-hamster transfection system to detect two cleavages in the 3' region of pre-18S rRNA. One cleavage occurred at the 3' terminus of 18S rRNA, whereas the other occurred approximately 55 nucleotides downstream of the mature 3' end of the 18S rRNA sequence in a G+U-rich region of the ITS1.…”