Small nuclear RNAs are encoded in the nontranscribed region of

Transcription

1 Proc. Natli cad. Sci. US Vol. 79, pp , May 1982 Biochemistry Small nuclear RNs are encoded in the nontranscribed region of ribosomal spacer DN (in vitro transcription/cloned rdn/southern analysis) RONLD REICHEL*, HNS-JURG MONSTEINt, HNS-WILLI JNSEN*, LENNRT PHILIPSONt, ND BERND-JOCHIM BENECKE** *Department of Biochemistry, Ruhr-University, D-63 Bochum, Federal Republic of Germany; and tdepartment of Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden Communicated by James E. Darnell, February 8, 1982 BSTRCT The structure of in vitro synthesized mouse small nuclear RN transcribed by RN polymerase I (snpi RN) was studied by T1 RNase digestion pattern analysis. The patterns of four different snpi RN species were different from those of the Ul and U2 RN species. In addition, the four different snpi RN species, ranging from to 2 nucleotides in length, yielded almost identical patterns. The snpi RN molecules hybridized to cloned mouse ribosomal DN containing the nontranscribed spacer DN and S ribosomal precursor RN molecules did not compete with this hybridization. Southern blot analysis of fragments from the ribosomal DN confirmed that snpi RN species exclusively hybridized to sequences corresponding to the so-called nontranscribed ribosomal spacer region. Initially, the existence of a class oflow molecular weight nuclear RN species that differ from the small nuclear RN (snrn) molecules by number and size was described in HeLa cells (1). These RN molecules can be labeled in isolated nuclei in vitro. Their synthesis is resistant to high concentrations of a-amanitin (2 g/ml) and they have therefore been called "small nuclear polymerase I RN (snpi RN)." HeLa cell snpi RN represents a large number ofdistinct RN molecules in the size range 6 S to 12 S with major products around 8 S. In contrast to ribosomal precursor RN, the synthesis of snpi RN is not inhibited by pretreatment of intact cells with low doses of actinomycin D, and subfractionation of nuclei seemed to indicate that the synthesis of snpi RN is in the nucleoplasm rather than in the nucleolar fraction. Cell fractionation studies in combination with short-term labeling kinetics demonstrated that snpi RN molecules also accumulate in vivo (1). snpi RN molecules have been detected in all mammalian cell lines studied. In contrast to other low molecular weight RN species, these molecules reveal a pronounced species specificity with an RN pattern unique to each animal species but similar in different cell types within a species (2). This remarkable species specificity may be used to separate evolutionarily related organisms such as mouse and rat or the closely related species gorilla and man. Because no detailed information is available with respect to snrn biosynthesis, including the question of whether or not these RN species are synthesized via precursor molecules, we wanted to investigate a possible relationship between snpi RN molecules and snrn species. Indirect evidence also suggested that RN polymerase I might be involved in snrn transcription (3). In the present report we compare in vitro synthesized mouse snpi RN species and two species of snrn (Ul and U2) by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. 173 solely to indicate this fact. 36 nuclease T1 oligonucleotide pattern analysis. The results show that snpi RN and these snrn species are not related to each other. Furthermore, the genes coding for snpi RN molecules have been localized within sequences of the nontranscribed ribosomal spacer region, immediately adjacent to the ' end of the S transcription unit. MTERILS ND METHODS In Vitro RN Synthesis. Nuclei were isolated from mouse 3T6 fibroblasts by lysis of the cells in hypotonic buffer (6 mm KCV1.6 mm MgCl2/i mm dithiothreitol/ mm Tris HCI, ph 8.) in the presence of.% Nonidet P-. The nuclei scraped off the dishes were washed in incubation buffer [1 mm magnesium acetate/i mm dithiothreitov.2 mm EDT/ mm Tris HCI, ph 7.9/2% (vol/vol) glycerol], resuspended, and preincubated with a-amanitin for min on ice. The final reaction mixture contained 1 mm TP, 1 mm GTP, 1 mm CTP, 2 um UTP, 2 mm MnCl2, mm magnesium acetate, mm (NH)2SO, 1 mm dithiothreitol, and 2,Ci (1 Ci = 3.7 X becquerels) of [3H]UTP (-6 Ci/mmol) or [a-32p]utp (>3 Ci/mmol) (mersham) in a total volume of 28,ul. Incubation was for 3 min at 2 C, and the RN was isolated after disruption ofthe nuclei with 1 ml of HSB (. M NaCl/ mm MgCl2/.1 M Tris HCI, ph 7.) and digestion ofthe DN with RNasefree () DNase I (,ug/ml; Boehringer Mannheim). The phenol-extracted RN was analyzed in 6-1% polyacrylamide gels as decribed (). Individual RN bands were eluted from the gel pieces electrophoretically in sealed dialysis bags coated with trn. T1 RNase products were separated by electrophoresis on cellulose acetate in the first dimension and homochromatography on DEE-cellulose in the second dimension, as described (6). Hybridization to Ribosomal DN (rdn). The cloning of the mouse rdn in bacteriophage and the characterization of the gtwes Mr 97 clone (obtained from I. Grummt, Wurzburg) have been described in detail (7, 8). In vitro synthesized snpi RN was hybridized to the cloned rdn under the following conditions: X SET (1 x SET is.1 M NaCl/l mm EDT/.3 M Tris HCl, ph 8.), X Denhardt's reagent (9), % (vol/vol) formamide,.1% NaDodSO, and Escherichia coli nucleic acids at 7 g/ml for 18 hr at 2 C. The filters were washed twice in X SET/. 1% NaDodSO, twice in 2x SET/.1% NaDodSO, and twice in.x SET/.1% NaDodSO at 2 C for min, respectively. Elution of the RN hybridized was at 8C for 3 min with sterilized water bbreviations: snrn, small nuclear RN; snpi RN, small nuclear polymerase I RN; rdn, DN containing the genes for ribosomal RN; kb, kilobase(s). * To whom reprint requests should be addressed.

2 Biochemistry: Reichel et al containing.% NaDodSO. Competition hybridization consisted ofprehybridization ofthe filters with unlabeled S rrn precursor (3 pug/ml) for 2 hr followed by incubation with labeled snpi RN for 18 hr as described above. To identify the sequences coding for snpi RN, the rdn ofclone Mr 97 was digested with Sal I and the fragments were separated in a.8% agarose gel. Transfer of the DN fragments to nitrocellulose filters was as described by Southern () and hybridization with 32P-labeled snpi RN was as above. The dried filters were exposed on Kodak X-Omat R films for days with a Cronex intensifier screen. Experiments involving recombinant DN were carried out under L2/B2 conditions in accordance with the German guidelines for recombinant DN research. RESULTS Isolated mouse 3T6 fibroblast nuclei were used for in vitro transcription studies in the presence ofvarious concentrations of a- amanitin. Low molecular weight RN species labeled in the presence of a-amanitin (2 jig/ml) (Fig. 1, lane 3) after pretreatment of the cells with low doses of actinomycin D to suppress transcription of ribosomal precursor RN selectively (11) have been designated "snpi RN molecules" (1, 2). These snpi RN molecules do not arise as a consequence of the pretreatment with actinomycin D because they also were observed in the absence of this inhibitor (Fig. 1, lane ), although the drug treatment resulted in enhanced synthesis of snpi RN molecules. When compared to the corresponding RN molecules obtained with HeLa cell nuclei (lanes 6 and 7), the mouse snpi RN pattern clearly revealed prominent RN species considerably smaller than the HeLa snpi RN species. The mouse snpi RN species migrated slightly differently from mouse snrn species labeled in vivo (lane 1). In order to determine whether the in vitro synthesized snpi RN molecules are related to the snrn species and therefore might represent the corresponding snrn precursors, T1 RNase digestion patterns of individual RN species from both RN populations were compared. The snrn U1 and U2 pat- Proc. Natl. cad. Sci. US 79 (1982) 37 terns (Fig. 2 and B) clearly differed from those obtained with snpi RN species 1, 2, 3, and, indicating that both RN populations do not represent common RN sequences. The snrn U1 and U2 patterns were similar to those published by other authors (12, ) but different from those obtained with snpi RN species. The simpler oligoribonucleotide pattern with snpi RN is explained by the fact that snpi RN molecules contain only one labeled nucleotide species (UMP). Furthermore, although differing in length by more than nucleotides, the four snpi RN species analyzed all reveal identical T1 RNase patterns, suggesting that they are composed of identical RN sequences. The mouse snpi RN species probably do not represent aggregates of the same molecules because reelectrophoresis of species 1- under denaturating conditions in gels containing 7 M urea (Fig. 1, lane ) still resulted in distinct RN bands with similar size differences. The patterns of longer snpi RN molecules did not reveal additional oligonucleotides, suggesting that snpi RN molecules are composed of repetitive nucleotide sequences. Because the snpi RN species are transcribed by the same RN polymerase that is involved in rrn transcription and because rdn is known to contain clusters of repetitive sequences associated with the nontranscribed spacer DN separating the individual rdn transcription units (1, 1), in vitro synthesized mouse snpi RN molecules were hybridized to cloned mouse rdn. The mouse rdn clone gtwes Mr 97, known to contain about two-thirds of the 18S rrn gene, the external transcribed spacer, and about 6.7 kilobases (kb) of the nontranscribed spacer ofmouse rdn (7, 8), was used as a DN source. snpi RN molecules hybridized efficiently to this rdn clone (Table 1). The hybridization of snpi RN was not inhibited by an excess of unlabeled S rrn precursor, although S rrn hybridization was inhibited effectively, suggesting that hybridization occurs outside the region corresponding to the S RN. It is interesting to note that HeLa cell snpi RN molecules did not hybridize to the mouse rdn clone. This is in good agreement with the finding that the "nontranscribed" ribosomal spacer shows extensive heterogeneity not only in length (1-17) but also in sequence (18-2) between K- LU L2- u2--,,o.i ~~ S U- U6- t-rn-._m_ _ 1 s_- 6 7 FIG. 1. Electrophoresis of in vitro synthesized low molecular weight nuclear RN. Lanes: 1, markers (mouse low molecular weight RN isolated from 3T6 cell cytoplasm after labeling with [3Hluridine at,ci/ml for 3 hr in vivo); 2, mouse nuclear RN synthesized in vitro in the presence of a- amanitin (,g/ml) after pretreatment of the cells with actinomycin D - (. ug/ml); 3, as 2 but in vitro syn- - thesis was in the presence of a-aman- -3 itin at 2 jg/ml;, in vitro synthesis -2 was with a-amanitin at,ug/ml but without pretreatment with actinomycin;, reelectrophoresis of mouse snpi RN bands 1- in 8% acrylamide gels containing 7 M urea; 6, HeLa cell snpi RN synthesized in vitro with a- amanitin ( jig/ml) and actinomycin D; 7, as 6 but in vitro synthesis was with a-amanitin at 2 jig/ml; 8, 32P-labeled mouse snpi RN synthesized in vitro as in 3; 9, mouse 2P-labeled snpi RN was hybridized to cloned rdn, recovered, and analyzed 8 9 as in 8.

3 38 Biochemistry: Reichel et al Proc. Natl. cad. Sci. US 79 (1982) la *1 I _b Sn RN ii_ :i'. C.: snpi RN ;...Ṗ,.is 1 a t * t r12 _ ~ ~ ~ ~ ~.,o *1)1 nrl, - nr2 B :1 p a W C: i k.fo tir 3 n' - electlopholesis FIG. 2. T1 RNase digestionpatterns analysis of mouse snrn and snpi RN. ( and B) snrn species U1 and U2 were isolated from nuclei after labeling of the cells with [32P]orthophosphate (2 pci/ml) in "low-phosphate" (6 mg/liter) Dulbecco's medium for 36 hr. (C-F) snpi RN species 1, 2, 3, and synthesized in vitro in the presence of a-amanitin (2,ug/ml) with 2 jci of [a-32p]utp per assay. Individual bands were cut from the gels, eluted electrophoretically, and analyzed after T1 RNase digestion. different animal species, analogous to results obtained with various mammalian snpi RN species (2). When 32P-labeled snpi RN was hybridized to DN isolated from gtwes Mr 97 clone and the RN recovered from the hybrids was analyzed by gel electrophoresis, a similar snpi RN pattern was obtained (Fig. 1, lanes 8 and 9). In order to determine the localization of sequences coding for snpi RN within the Mr 97 clone, 32P-labeled snpi RN was hybridized to DN fragments generated by Sal I. The restriction map of gtwes Mr 97 as elaborated by Grummt et al (7, 8) is presented in Fig. 3. The two Sal I sites in the right arm and the four sites in the Table 1. Hybridization of snpi RN to cloned mouse rdn Hybridization probe cpm Hybridization probe cpm Mouse snpi RN 3,69 Mouse S rrn precursor 33 Mouse snpi RN + Mouse S rrn precursor unlabeled S + unlabeled S rrn rrn precursor precursor (3 pg/ml) 77 (3,ug/ml) 2,978 Mouse S rrn precursor HeLa snpi RN 17 (blank filter, loaded with Mouse snpi RN E. coli DN) 8 (blank filter) 1 3H-Labeled snpi RN (, cpm in a total volume of 3 /) or 32P-labeled S rrn (3, cpm) was hybridized to duplicate filters loaded with gg of mouse Mr 97 DN. insert will lead to a shortened right arm (12.6 kb), the left arm (23.2 kb) carrying about 1,9 nucleotides of the 18S gene, a.8-kb fragment from the right arm, and four additional fragments- (.7 kb), B (3.3 kb), D (1.7 kb), and E (.7 kb)- representing external transcribed and nontranscribed ribosomal spacer sequences. In Fig., lane shows the expected Sal I restriction fragments, B, and D in addition to the left and right arms. The two smaller fragments,.7 and.8 kb, were not detectable because this region of the gel is obscured by a smear of low molecular weight material. This material originates from contaminating E. coli nucleic acids because in this experiment the phages were purified by only one cesium chloride centrifugation step. snpi RN hybridized only to the 3.3-kb Sal I fragment B consisting of about 6 nucleotides of nontranscribed ribosomal spacer and about 2.7 kb ofexternal transcribed spacer (Fig., lane B). Nucleotide, sequence analysis of the initiation region of the ribosomal transcription unit from mouse has revealed a Pvu II site upstream from the ' end of the S rrn precursor at position -17 (8). ccordingly, ifpurified Sal I fragment B is digested with Pvu II, two fragments can be resolved, a smaller one of61 nucleotides consisting entirely ofnontranscribed spacer sequences and a larger one of about 2.8 kb including most of the external transcribed spacer and the preceding 16 nucleotides of nontranscribed spacer DN (Fig., lane C). Hybridization to the cor-

4 Biochemistry: Reichel et al Proc. Natl. cad. Sci. US 79 (1982) 39 W E- 23,2 Sal I site? Pvu site,7 3,3 1,7 1 I i,7,8 IS 12,6 r DN insert 11,3kb N c Pvu E Sal I-B FIG. 3. Restriction map of the gtwes Mr 97 mouse rdn clone as elaborated by Grummt et al. (7). rrows indicate the location of the Sal I cleavage sites. The Pvu II site within the expanded 3.3-kb Sal I fragment is shown at the bottom. 3,3kb 1 responding Southern blot demonstrated that both fragments contain DN sequences complementary to snpi RN (Fig., lane D). Hybridization was not due to some random base pairing because treatment of the hybrid filters with pancreatic RNase (Fig., lane E) had no effect. In order to demonstrate that snpi RN does not hybridize to the external transcribed spacer sequence, blots were prehybridized with a large excess (3,tg/ ml) of S rrn precursor. This did not prevent hybridization of snpi RN to either fragment (Fig., lane F). Therefore, hybridization to the 2.8-kb fragment containing the external transcribed spacer must be due to the 16-nucleotide sequence of nontranscribed spacer DN within this fragment. Together, these results demonstrate that snpi RN molecules are transcribed from the so-called nontranscribed ribosomal spacer of the mouse rdn. kb e M B DISCUSSION The structure of in vitro synthesized mouse snpi RN molecules has been analyzed by T1 RNase digestion patterns and these patterns were compared to the oligonucleotide patterns obtained with snrn species U1 and U2 ofabout the same size. It is shown that these classes ofsnrn molecules are not related to each other. This is also true for snrn U3 because HeLa cell U3 RN does not hybridize to a HeLa rdn clone containing snpi RN genes (unpublished data). Conversely, the failure of HeLa cell snpi RN to hybridize to cloned snrn genes isolated from a human gene library in our laboratory (unpublished data) also rules out a relationship between these classes of low molecular weight RN. To date, snpi RN molecules are characterized by three remarkable findings: (i) these molecules are synthesized by RN polymerase I, the enzyme involved in ribosomal precursor RN synthesis; (ii) snpi RN molecules are transcribed from repetitive DN sequences; and (iii) these low molecular weight nuclear RN species reveal an extensive divergence between different animal species, even among evolutionarily related organisms. The latter two properties are shared by the external kb B C D E F 'Os FIG.. Separation of restrictionfragments of clone Mr 97 in.8% agarose gels and hybridization with snpi RN. Lanes: M, marker digested with Hindu;, Sal I restriction fragments of Mr 97; B, hybridization of 32P-labeled in vitro synthesized mouse snpi RN to Southern blots of. FIG.. Hybridization of snpi RN to Southern blots of the 3.3-kb Sal I fragment digested with Pvu H. Lanes:, marker digested with HindI; B, reelectrophoresis of the 3.3-kb Sal I fragment of Mr 97; C, 3.3-kb Sal I fragment digested with Pvu II; D, hybridization of snpi RN to Southern blots of the Pvu II fragments; E, as D but the hybridized filters were treated with pancreatic RNase (,ug/ml) for 3 min at 37 C; F, as E but hybridization was in the presence of S rrn precursor (3 yg/ml).

5 31 Biochemistry: Reichel et at nontranscribed spacer sequences associated with the ribosomal genes (1-22). In addition, the involvement of RN polymerase I further points to a possible relationship of snpi RN transcription and ribosomal genes. These findings prompted us to look for sequence complementarity between snpi RN and rdn including nontranscribed spacer sequences. n appropriate mouse rdn clone (gtwes Mr 97) has been isolated and extensively characterized by Grummt et al (7, 8). The results presented here demonstrate that nontranscribed spacer sequences immediately adjacent to the ' end ofthe S transcription unit indeed code for snpi RN molecules. The DN sequence of this region has been determined recently (8). From this it can be predicted that the predominant spots and 6 in Fig. 2 correspond to the sequences [C-U]G and [C-U-U]G, respectively, which are present in 9 and 7 mol per 3 nucleotides on the ' side of the S rrn gene. The patterns thus support our idea that the snpi RN is unrelated to other snrn genes including the gene for U3 RN (23) which contains only 2 mol of each of these oligonucleotides. The snpi RN molecules must be rapidly exported from the nucleolus because nuclear fractionation studies as well as electron microscopic autoradiography showed an almost exclusive association of snpi RN with the nucleoplasmic fraction of HeLa cells (1). Our finding that parts of the so-called nontranscribed ribosomal spacers are actively transcribed is in good agreement with electron microscopic studies by Scheer et al (2, 2) who observed transcription complexes and nascent RN chains associated with nontranscribed spacer regions of Xenopus oocytes. In addition, by using cloned rdn exclusively containing nontranscribed spacer sequences, Rungger et al (26) identified RN transcripts complementary to the central parts of the nontranscribed rdn spacer in cultured Xenopus epithelial cells. These transcripts were found to represent a heterogeneous RN population with a size between and 23 S. HeLa cell snpi RN and mouse snpi RN molecules have similar size distributions when analyzed in sucrose gradients (ref. 1; unpublished data). However, it is not clear at present whether the RN molecules identified in Xenopus cells and the snpi RN decribed here belong to the same class of RN molecules because, up to now, the synthesis and structure of the former RN population have not been characterized biochemically. In addition, in contrast to the results obtained by Rungger et al, even with the larger snpi RN molecules we do not observe any hybridization to sequences of the nontranscribed rdn spacer further away from the S transcription unit-for example, Sal I fragment D (not shown). The most intriguing question remaining concerns the physiological role of snpi RN molecules. One possible explanation could be that, instead of using an initiation site at the ' end of the S transcription unit, initiation of RN polymerase I also takes place at various sites upstream from this position and snpi RN molecules are generated by cleavage of such a putative rrn pre-precursor molecule during a very early processing step. The postulation of new initiation sites for polymerase I is consistent with the failure to detect a '-terminal triphosphate on mammalian S rrn precursor molecules (27, 28). In addition, Bach et al (8) found only a minor fraction of S rrn precursor (about -1%) capable of accepting a cap structure supplied by the vaccinia virus capping enzyme in vitro. This indicates that only a limited amount of rrn precursor molecules are bearing a polyphosphate at the ' terminus. The additional initiation sites for RN polymerase I postulated here might explain why, after inactivation of the ribosomal genes by low doses of actinomycin D, increased amounts of snpi RN molecules accumulate, possibly as a result of a shift ofthe major Proc. Nad cad. Sci. US 79 (1982) initiation site to a position (or positions) located upstream in the nontranscribed ribosomal spacer. nother possibility could be that snpi RN molecules are transcribed independently and are involved in rrn precursor processing, comparable to the role of snrn in the processing of heterogeneous nuclear RN. Finally, it should be mentioned that snpi RN genes are not representing a class of reiterated sequences dispersed throughout the genome-for example, like thelu family sequences-because restriction ofgenomic DN and hybridization of snpi RN to the DN fragments shows only a single DN band hybridizing to snpi RN. 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