Cell, Vol. 60, 697408,

March

23, 1990, Copyright

0 1990 by Cell Press

The U3 Small Nucleolar Ribonucleuprotein Functions in the First Step of Preribosomal RNA Processing Susan Kass: Kaximierz lyc,t Joan A. Steitz,t and Barbara Sollner-Webb’ Human Genetic8 Program and Biological Chemistry Department The Johns Hopkins University School of Medicine Baltimore, Maryland 21205 t Department of Molecular Biophysics and Biochemistry Howard Hughes Medical Institute Yale University School of Medicine New Haven, Connecticut 06510 l

Summary The first cleavage in mammalian pre-rRNA maturation occurs near the 5 end within the 5’ external transcribed spacer. Using mouse cell extracts, we show that this processing is abolished by micrococcal nuclease pretreatment. Autoantibodies that recognize the U3, U8, and U13 snRNPs (anti-fibrillarin) deplete processing activity from the extract and selectively immunopmcipttate both rRNA substrates and processing products from the reactfon. Specific involvement of the U3 snRNP is demonstrated by native gel electtophoresis of the processing reaction followed by Northern blotting and by olfgonucleohdedimcted RNA ase l-l abolition of processing activity. Our ldentification of U3 function is discussed with respect to the molecular basis of pre-rRNA recognition by the U3 snRNP, possible roles of U3 and other nucleolar snRNPs in rRNA processing, and the morphological organixation of the nucleolus and the ribosomal transcription complex. Introduction The nucleolus of all eukaryotic cells is devoted to the synthesis, processing, and assembly of rRNA into ribosomes. In mammalian cells, rRNA is transcribed as a ~475 precursor (Tiollais et al., 1971), which is subsequently cleaved in multiple steps to yield the mature 18S 5.8S, and 28s rRNA species; the various “transcribed spacer sequences; which comprise over half of the primary transcript, are rapidly degraded (reviewed in Perry, 1976; Fedoroff, 1979; Bowman et al., 1983; Crouch, 1984; Mandal, 1984). However, the processing sites are not located solely at the borders of the mature rRNAs; indeed, the first processing event that mouse pre-rRNA undergoes is cleavage at residue ~650 (3.5 kb upstream of the 18s sequences) with a concomitant degradation of the upstream fragment (Miller and Sollner-Webb, 1981; Gurney, 1985; diagrammed in Figure IA). This “primary processing” event occurs so rapidly in vivo that the 5’ end of the predominant pre-rRNA (the “45s pre-rRNA”) maps to this site. An analogous processing event occurs in the 5’ external transcribed spacer

of human pm-rRNA at position ~414 (Kass et al., 1987) and virtually the same processing signal is found in rat pre-rRNA starting at position 790 (Rothblum et al., 1982; Bourbon et al., 1988; Stroke and Weiner, 1989). A related sequence is also found in the Xenopus external transcribed spacer (Mougey et al., unpublished data). The primary rRNA processing reaction has been successfully reproduced in extracts of cultured mouse cells, both in coupled transcription-processing systems (Miller and Sollner-Webb, 1981) and using exogenously added in vitro synthesized rRNA substrate (Kass et al., 1987). Mimicking the in vivo situation, the upstream cleavage product is rapidly degraded. Until very recently (Hannon et al., 1989) this has been the only metazoan rRNA processing event that could be reproduced in vitro. Interestingly, human as well as mouse pre-rRNA substrates are processed in the mouse in vitro system. This is consistent with the findings that the 200 nucleotides just 3’to the processing site are sufficient for processing (Craig et al., 1987) and that this region is strikingly conserved between mammals (Kass et al., 1987) while the surrounding sequences have completely diverged. Using nondenaturing polyacrylamide gel electrophoresis, we have recently found that factors of the cell extract form a specific complex with rRNA substrates that are competent to undergo primary processing, but not with RNAs from which essential processing signals have been deleted (S. K. and B. S., unpublished data). Complex formation is competed by processing-competent but not by processing-incompetent rRNA, and the complex remains stably attached to both the pre-rRNA and the downstream processed product. Six polypeptides have been detected by UV cross-linking to be specifically associated with the rRNA in the processing complex. In the last several years, small nuclear ribonucleoprotein particles (snRNPs) have been shown to be of fundamental importance in RNA maturation, both in premRNA splicing (the nucleoplasmic Ul, U2, and U4-U6 snRNPs; reviewed in Maniatis and Reed, 1987; Steitz et al., 1988b) and in 3’end maturation of histone mRNA (the U7 snRNP; reviewed in Birnstiel and Schaufele, 1988; Mowry and Steitz, 1988). We therefore considered whether nucleolar snRNPs might be involved in rRNA processing. The best known and most abundant nucleolar snRNP is the U3 RNP (about 2-10 x 105 copies per mammalian cell; reviewed in Reddy and Busch, 1988). Human U3 RNP contains at least six proteins (Parker and Steitz, 1987) and a 217 nucleotide RNA (Suh et al., 1986), which is highly conserved across a wide range of eukaryotic organisms. One of the proteins is the 34 kd “fibrillarin,” which is the target of certain scleroderma patient autoantibodies (Lischwe et al., 1985; Ochs et al., 1985). Recent analyses of anti-fibrillarin immunoprecipitates from human cells have revealed the existence of several additional small nucleolar RNPs (Tyc and Steitz, 1989). The RNAs of two of these, called U8 and U13, possess a 5’trimethyl guanosine cap residue, as does U3, but are less abundant than

Cell 898

The U3 snRNP has been suggested to play a variety of roles in the processing pathway of rRNA, based on observations that U3 is complementary to, hydrogen-bonded with, able to be cross-linked to, or immunoprecipitated with various regions of the pre-rRNA transcript (Prestayko et al., 1970; Calvet and Pederson, 1981; Crouch et al., 1983; Bachellerie et al., 1983; Epstein et al., 1984; Tague and Gerbi, 1984; Parker and Steitz, 1987; Tollervey, 1987; K. Parker et al., 1988; Kupriyanova and Timofeeva, 1988). These models include interaction of U3 at the boundary between the 5.8s region and the second internal transcribed spacer, at the 3’ end of 28S, or upstream of that site within the 28s region. Recently, in vivo psoralen cross-linking studies have localized a U3-rRNA contact to the 5’ transcribed spacer region within a few hundred nucleotides of the primary processing site in human (Maser and Calvet, 1989) and rat (Stroke and Weiner, 1989) pre-rRNA. The human cross-link maps between nucleotides 438-895, just downstream of the processing site at position ~414, while the rat cross-link maps within the region 767’1149, surrounding the processing site (position m790) in that species. Stroke and Weiner pinpointed the cross-linked residues in U3 RNA near the 5’ end of the molecule, adjacent to the most highly conserved region of U3, known as box A. Our studies, using the mouse in vitro primary processing reaction and a variety of approaches to test U3 involvement, extend and confirm the in vivo cross-linking results. We find that the U3 snRNP not only binds selectively to rRNA processing substrates and their downstream cleavage products, but also is required for the primary processing reaction itself.

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(A) Above is shown a map of the mouse rRNA coding region and surrounding spacer regions; the -850 processing site is indicated by the vertical arrow. The middle panel shows an expansion of the -650 processing region, with the 200 nucleotide mammalian conserved segment represented by hatching. The bottom panel diagrams T7 RNA polymerase-synthesized rRNA substrates, beginning with 28 nucleotides of T7 promoter/polylinker sequence and containing rRNA sequences extending from the indicated position to residue 1290. (B and C) Ceil extract was treated with micrococcal nuclease (MN) or was mock treated (-) in the presence of 1 mM CaCls. EGTA (E; 3 mM) was added at the indicated times (in minutes), as was tRNA, total yeast RNA, or poly(ACI). Then =P-labeled rRNA substrate (RNA 312-1290) was added, and following a 45 min reaction period, RNA was isolated and electrophoretically resolved.

U3. Hence, mammalian nucleoli contain at least one family of related snRNPs as well as other small RNA-protein complexes targeted by other autoantibodies (e.g., antiTh [Hashimoto and Steitz, 1983; Gold et al., 19891 and anti[5S]RNP [Steitz et al., 1988a]). In yeast, the U3 analog snR17 is essential for cell viability (Hughes et al., 1987).

Primary rRNA Processing Requires a Cellular RNA Component The primary processing of a mouse pre-rRNA substrate requires cellular components present in S-100 or Dignamtype extracts of mouse cells (Miller and Sollner-Webb, 1981). Although it was originally assumed that these components were entirely proteinaceous (Kass et al., 1987) we noted that a recently characterized rRNA processing complex (S. K. and B. S., unpublished data) had greatly retarded electrophoretic mobility in nondenaturing gels, similar to mammalian splicing complexes (Konarska and Sharp, 1986). We therefore investigated whether a cellular nucleic acid species might be important for the primary processing by incubating active S-100 extract (Figure lB, lane 2) with micrococcal nuclease in the presence of CaC12. After inactivation of the nuclease with EGTA, the processing activity was assayed by addition of radiolabeled rRNA substrate (diagrammed in Figure lA, bottom). Processing was not detectable in the nuclease-treated extract (Figure lB, lane 5); the substrate rRNA was unchanged relative to the unincubated material (lane 1). Controls demonstrated that the processing activity was not diminished in extracts that had been pretreated with the calcium-containing processing buffer in the absence of micrococcal nuclease (lane 3) or in extracts that had

U3 RNP Functions 899

in Pre-rRNA

Processing

i;i E

rRNA primary processing reaction. Figure 1C shows that several different RNAs, added in various amounts, did not restore processing activity (lanes 5-10) to an extract that was inactivated by pretreatment with microcoocal nuclease (compare lane 4 with lanes 2 and 3). Thus, a specific RNA component present in the cell extract is most likely required for processing.

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Figure 2. lmmunodepletion by Anti-Fibrillarin Antibody

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8 in the S-100 Extract

S-100 extracts were depleted by three rounds of immunoprecipitation with anti-fibrillarin antibodies (lanes 5-7) nonimmune human serum (lane 3) monoclonal anti-Sm antibody (lane 4) or antiTh human serum (lane 8). The mock immunoprecipitation reaction (lane 2) did not contain any antibody. RNA was isolated from aliquots of both precipitates and supernatants, fractionated on the same 10% denaturing polyacrylamide gel, and probed for U3 by Northern blot hybridization. Autoradiographs were quantitated by densitometry. Aliquots of the depleted extracts were also assayed for the processing activity using the 3121290 rRNA precursor shown in lane 1.

been incubated with the nuclease in the absence of Car& (lane 4). We conclude that a nucleic acid component of the extract is important for the primary rRNA processing reaction. Since micrococcal nuclease can digest DNA as well as RNA under the low salt conditions used in our assays, we repeated the experiment of Figure 16 using DNAase I instead of micrococcal nuclease. DNAase I pretreatment did not impair the processing capacity of the extract, even when 25 times more enzyme was used than is sufficient to eliminate an added tracer double-stranded DNA (data not shown). This strongly suggests that it is an RNA rather than DNA component of the extract that is important in the primary processing. Certain in vitro RNA processing systems may require the presence of nonspecific RNA (Ryner and Manley, 1987) but this does not appear to be the case with the

Evidence for the Involvement of a Nucleolar snRNP To investigate whether the required RNA-containing component might be a nucleolar snRNR we used anti-fibrillarin antibodies, specific for U3, U8, and U13 particles, to deplete the active processing extract (Figure 2). Depletion was assessed by monitoring the level of U3 RNA remaining (Figure 2, top and middle). Three rounds of immunoprecipitation of the S-100 extract either with monoclonal anti-fibrillarin (lane 5) or with scleroderma patient antibodies (lanes 6 and 7) resulted in about 80% removal of U3, consistent with previous observations that this snRNP is difficult to immunoprecipitate quantitatively (Parker and Steitz, 1987); control anti-Sm (which targets the Ul, U2, U4/U6, and U5 snRNPs [Lerner et al., 19811) or antiTh (which targets the Th[7-2) and RNAase P particles [Hashimoto and Steitz, 1983; Gold et al., 19891) antibodies had no effect on the level of U3 (lanes 4 and 8). Those extracts exposed to anti-fibrillarin antibodies reproducibly exhibited substantially diminished processing activity (Figure 2, bottom; compare lanes 5-7 with lane 2), while those treated with nonimmune or control antisera retained full activity (lanes 3, 4, and 8). Hence, it seemed likely that a fibrillarin-containing snRNP is required for primary processing. In these immunodepletion experiments, the diminution of processing activity is less than the extent of antigen precipitation (only 2- to 3-fold loss of activity compared with 5-fold U3 depletion in the experiment of Figure 2). Several reasons for this are conceivable, including the possibility that the anti-fibrillarin precipitated component is not the limiting factor in the S-100 extract. Consistent with this suggestion, the processing capacity of the normal S-100 extract can be stimulated about 2- to 3-fold when it is supplemented with an inactive, micrococcal nuclease-treated extract (data not shown). Intact U3 snRNA Is Required for Primary rRNA Processing To determine which snRNP in the U3, U8, U13 family might be involved in the primary processing reaction, we undertook oligonucleotide-directed RNAase H targeting experiments. We focused on the U3 particle for several reasons it is the most abundant nucleolar snRNP (Tyc and Steitz, 1989); unlike U8 and U13, it efficiently fractionates into mouse S-100 extracts (data not shown); and recent psoralen cross-linking results suggest that it may be associated with rRNA in the vicinity of the primary processing site (Maser and Calvet, 1989; Stroke and Weiner, 1989). Moreover, the susceptibility of U3 to RNAase H attack had been previously characterized (Parker and Steitz, 1987). As reported for human U3, we find that the mouse U3 particle is relatively resistant to digestion, even when a variety of

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Figure 3. Processing Activity in S-100 Extracts Pretreated with RNAase H and US-Specific Oligonucleotides Extracts were pretreated with RNAase H in the presence of US-specific oligonucleotides (lanes 5-12) or control U2-L15 (lane 3) or U118 (lane 4) oligonucleotides. U1/8 targets both Ul and U8 snRNPs in humans; its ability to target mouse U8 cleavage has not been analyzed. The samples were then divided into two parts. In (A), RNA was isolated from one part, fractionated in a 10% denaturing polyacrylamide gel, and probed for U3 RNA by Northern blot hybridization. The same blot was then reprobed for U4 RNA to control for loading. In (B), the other part of each sample was assayed for processing activity using the 312-1290 rRNA precursor. Lane M in (A) stands for DNA markers, whose lengths are indicated on the left. Specificitiesof the oligonucleotides are shown on the top of each panel. (C)The sequence of mouse U3B RNA was taken from Mazan and Bachellerie (1988). Evolutionarily conserved sequences, called boxes A, B, C, and D, are shaded. Oligonucleotides complementary to the marked sequences were used for the RNAase H experiments of (A) and (B).

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complementary oligonucleotides (diagrammed in Figure 3C) are tested (Figure 3A, lanes 5-12). However, one oligonucleotide complementary to U3 residues 64-79 elicits efficient cleavage of the U3 RNA in the extract (lane 7) with-

out altering 3A, bottom).

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U3 RNP Functions 901

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was efficiently cleaved showed markedly reduced activity (Figure 38, lane 7); all the other treated extract samples, which retained the bulk of the U3 intact, catalyzed efficient processing (lanes 2-6 and 8-12). These include control extracts treated with oligonucleotides that direct cleavage specifically of U2 (Figures 3A and 38, lane 3) or Ul (lane 4) snRNA. Thus, the integrity of U3 appears critical for processing activity. The U3 snRNP Is Part of the rRNA Processing Complex To examine whether the U3 snRNP is complexed with the substrate rRNA during primary processing, we incubated radiolabeled rRNA with S-100 extract under processing conditions and then immunoprecipitated with anti-fibrillarin antibodies. As shown in Figure 4A, both the 312-1290 (top) and 645-1290 (middle) precursors and their respec-

15

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5 of rRNA-snRNP Primary Pro-

(A) Radiolabeled 312-1290, 645-1290, or 669-1290 rRNA substrates (lane 1) were incubated with S-100 extract in processing buffer at WC or o”c, as indicated. Then the reaction mixtures were precipitated with nonimmune human serum (lanes 3 and 11) monoclonal anti-Sm antibody (lanes 4 and 12) monoclonal anti-fibrillarin antibody (lanes 5, 9, 13, and 16) human anti-fibrillarin serum (lanes 6, 10, 14, and 17) or anti-Th serum (lanes 7 and 15). (6) Radiolabeled 312-1290 rRNA substrate was incubated at 30°C in S-100 extract pretreated in the absence (lanes 1,2. and 4) or presence (lanes 3 and 5) of micrococcal nuclease for 30 min and precipitated with the monoclonal 7269 anti-fibrillarin antibody. RNA was isolated from both precipitates and supernatants and analyzed in a 4% denaturing poly acrylamide gel.

tive downstream cleavage products are precipitated by monoclonal as well as patient anti-fibrillarin antibodies. This is demonstrated by the appearance of precursor and product rRNA in the pellet (lanes 5 and 6) and their depletion from the supernatant (lanes 13 and 14). No precipitation was obtained using nonimmune serum (lane 3) or antibodies directed against other abundant snRNF+s present in the extract (lanes 4 and 7). These results indicate a relatively stable interaction between a fibrillarincontaining snRNP and the pm-rRNA in the processing complex. The very efficient precipitation of the downstream cleavage product further suggests that the snRNP remains attached even after the cleavage reaction is complete. The immunoprecipitation data of Figure 4A also show that the interaction of the antigenic snRNP with the rRNA substrate exhibits the same requirements as the processing reaction itself. First, binding is dependent on the pres-

Cell 902

U3 probe

B

Figure 5. Analysis of U3 RNA in Electrophoretically Separated Complexes

rRNA probe

Processing reactions containing the indicated RNAs (rRNA substrates [312-1290 and 64512901 and processing incompetent RNAs [rRNA 669-1290 and a 900 nucleotide pGEM transcript]) were incubated and then either treated or not treated with heparin. They were electrophoretically resolved on nondenaturing gels, blotted, and analyzed with the following probes: (A) a probe for U3 sequences; (6) a probe for rRNA sequences 645-1290; this is a reprobing of the blot of part (A); (C) a probe for U2 sequences; (D) a probe for U3 sequences; the reactions for (D) were incubated at 4%, not at 30%. The solid arrows represent the positions of specific shifted complexes; the hollow arrows represent the positions to which specific shifted complexes migrated in control lanes on the same gel.

C

U2 probe

D

U3 probe (reactIons

ence of a functional processing signal in the rRNA. The substrate extending from residue 645 to 1290, which is as efficiently processed as the longer 312-1290 substrate (Craig et al., 1987) is precipitated identically (Figure 4A, middle) to the 312-1290 molecule. In contrast, an rRNA containing only residues 669-1290, which does not support processing (Craig et al., 1987) is not precipitated by the anti-fibrillarin antibodies (bottom). Second, specific immunoprecipitation of the substrate, like processing, does not occur at 0% but requires incubation at 30% (Figure 4A, lanes 8-10). Third, binding of the snRNP to the processing substrate is abolished upon pretreatment of

Incubated

at 4=9

the S-100 extract with micrococcal nuclease (Figure 48, lanes 3 and 5) as is the processing activity (Figure 1). This latter result (Figure 48) further shows that it is not simply the binding of free fibrillarin protein that is responsible for the selective precipitation of processing-competent rRNAs in Figure 4A. The RNAase H data of Figure 3 strongly suggest that it is the U3 particle, the most abundant of the nucleolar snRNPs, which interacts with the rRNA substrate as detected in Figure 4. To verify this supposition, we used RNA mobility shift analysis, shown in Figure 5. After incubation of various unlabeled processing-competent or process-

U3 RNP Functions 903

in Pre-rRNA

Processing

ing-incompetent RNAs in the S-100 extract, the resulting nucleoprotein complexes were resolved on a nondenaturing polyacrylamide gel and analyzed by Northern blotting. The substrates were rRNAs extending from residues 312 to 1290 or from 645 to 1290, both of which are efficiently processed at position ~650 (Craig et al., 1967) and form specific, slowly migrating complexes competed by processing-competent but not by processing-incompetent RNAs (S. K. and 9. S., unpublished data). Negative control transcripts included an rRNA extending from residues 669 to 1290 and a -900 nucleotide pGEM transcript, both of which form only “nonspecific” complexes that migrate rapidly and are not selectively competed by processing-competent RNAs (S. K. and 9. S., unpublished data). Figure 5A shows the results obtained when blots of the electrophoretically separated nucleoprotein complexes were analyzed using a probe complementary to U3 RNA. In addition to rapidly migrating material (which we assume to be uncomplexed U3 snRNPs since it is observed under all conditions), a slowly migrating complex appears specifically in those lanes containing processing-competent RNAs (lanes 2-3 and 7-6). The slowly migrating U3-containing complex is not detected with the two control RNAs, which are not substrates for the processing reaction (lanes 4-5 and 9-10). Furthermore, the inclusion of low amounts of heparin (Figure 5A, lanes 6-10) does not alter the substrate specificity of formation of the retarded U3-containing complexes; it does, however, increase their mobilities somewhat, presumably because of removal of loosely bound components. When the same blot was reanalyzed with a probe complementary to the input rRNA (Figure 5B), the bulk of the processing-competent substrate is seen to comigrate with the retarded U3 snRNP. Strikingly, this is true both in the absence and the presence of heparin; heparin shifts the rRNA signal to coincide with the shifted U3 signal (Figure 59, lanes 2-3 and 7-6; compare with Figure 5A). In contrast, the processing-incompetent rRNA starting at residue 669 forms only a heterogeneous smear of more rapidly migrating material (Figure 59, lanes 4 and 9) that does not overlap the U3 signal (Figure 5A, lanes 4 and 9), even though this rRNA differs from the processing-competent RNA by only 24 out of ~650 nucleotides. Furthermore, the -900 nucleotide pGEM transcript (which migrates somewhat slower than the ~650 nucleotide rRNA of lanes 4 and 9; data not shown) also does not cause the retardation of U3 RNA (Figure 5A, lanes 5 and 10). (The strong rRNA signal at the top of each gel lane is evidently due to prerRNA present in the cell extract since it appears in the “no RNA” lanes as well.) The specificity of the U3 shifting strongly indicates that the retarded complexes at the positions of the solid arrows in lanes 2, 3, 7, and 6 (Figure 5A) represent active processing complexes. Since these reactions were performed under conditions of limiting rRNA substrate, it is not surprising that processing-competent rRNAs are shifted into the active processing complex quantitatively (Figure 5B), while only a fraction of U3 is shifted (Figure 5A). In fact, titration experiments indicate that each microliter of extract can cause specific gel retardation of ~0.1 pmol of rRNA. This implies

an estimated 2 x 10” U3 molecules per pl of extract assuming that U3 is in roughly 3-fold excess relative to the limiting processing component of the extract (data not shown; see above), in close agreement with the calculated abundance of U3 (Prestayko et al., 1970) of k2.5 x 10” molecules per pl of cell extract. The specific association of the U3 snRNP and the rRNA substrate in a processing complex is further verified by the experiments of Figures 5C and 5D. First, a blot like that of Figure 5A was stripped after probing for U3 and reprobed for U2 snRNA; no signal was seen at the position of the U3-rRNA complexes (the hollow arrows of Figure 5C), indicating that the rRNA substrate does not merely “stick” to any snRNl? Furthermore, the specific retardation of U3 requires that the reaction mixture be incubated at 30% (Figure 5A) and does not occur after incubation at 4% (Figure 50; the hollow arrows indicate the position expected for the retarded U3-rRNA complex). Primary rRNA Processing Does Not Require Pyrophosphate Bond Energy Since mRNA splicing requires ATP both to assemble snRNPs and the pre-mRNA into a spliceosome and to carry out the splicing reaction (Sharp, 1987; Steitz et al., 1988b), we investigated whether the snRNP-requiring primary rRNA processing event also uses pyrophosphate bond energy from an NTI? We treated the S-100 extract exhaustively with bacterial alkaline phosphatase to deplete its endogenous pool of ribonucleotides (and deoxyribonucleotides) (Wilkinson et al., 1983) and verified complete digestion by adding a tracer [a-32P]CTP to the reaction (Figure 69; even a lo-fold longer exposure revealed no residual CTP or CDP in lane 3). Upon addition of substrate, this extract was found to process at least as efficiently as the control, untreated extract (Figure 6A). Thus, it appears that the primary rRNA processing event does not require high energy phosphate bond cleavage, suggesting that complex snRNP-snRNP interactions like those involved in splicing may not be necessary. Discussion We have established that the most abundant snRNP of the mammalian nucleolus, which contains U3 RNA, participates in the earliest cleavage event of pre-rRNA processing. An RNA was shown to be essential for this processing by nuclease digestion experiments (Figure l), and a particle belonging to the newly described U3, U8, and U13 class (Tyc and Steitz, 1989), all of which share the 34 kd autoantigen fibrillarin, was implicated by immunodepletion and immunoprecipitation experiments (Figures 2 and 4). Inhibition of in vitro processing by RNAase H-mediated cleavage of U3 RNA (Figure 3) and the coordinate retardation of U3 and the rRNA substrate upon native gel electrophoresis (Figure 5) then identified the U3 snRNP as specifically involved. Not only does the U3 snRNP bind specifically to the processing substrate, but it remains bound to the processed rRNA product (Figure 4). This is the first direct evidence that a snRNP functions in rRNA processing. Thus, all of the highly abundant (>18 copies

U3 RNP Functions 905

in Pre-rRNA

Processing

event to rRNA biogenesis remains mysterious, efficient processing of the 5’ external transcribed spacer is very widespread in nature and thus must confer a substantial selective advantage. The primary processing event occurs in all mammals examined, where the processing signal is highly conserved (mouse [Miller and Sollner-Webb, 19811; human [Kass et al., 19871; rat [Stroke and Weiner, 19891) in contrast to flanking spacer sequences, which are completely diverged. In addition, 5’ processing appears to occur in other vertebrate rRNAs (e.g., Xenopus) where a related sequence is present (Mougey et al., unpublished data) as well as in organisms as diverse as Neurospora (Tyler and Giles, 1985), Physarum (Blum et al., 1986), Tetrahymena (Sutiphong et al., 1984), wheat (Barker et al., 1988), Artemia (Koller et al., 1987), and Bombyx (Fujiwara and Ishikawa, 1987), where there is no obvious sequence homology. The mammalian primary processing event is evidently not essential for at least some of the subsequent rRNA processing steps. Cleavage at the 5’ border of 18s sequences can occur on artificial substrates lacking the primary processing site both in vivo (Vance et al., 1985) and in vitro (Hannon et al., 1989); and in the Xenopus oocyte, 18s and 5.8s processing can occur even after efficient cleavage of endogenous U3 snRNA induced by an oligonucleotide analogous to that used successfully in Figure 3 (Savino and Gerbi, 1989). Hence, the primary processing event may serve to asssemble a U%containing complex that is stimulatory rather than essential for subsequent rRNA maturation steps, such as the next processing event, which liberates the 3’end of the 28s region (Gurney, 1985; Parker and Steitz, 1987) or the later cleavages that release 5.8s rRNA (Crouch et al., 1983; Bachellerie et al., 1983; Tague and Gerbi, 1984). The U3 snRNP could also contribute to large ribosomal subunit assembly since an in vitro interaction between U3 and a conformationally important internal region of 28s RNA near the a-sarcin site has been detected (K. Parker et al., 1988). The existence of yeast U3 RNA (Hughes et al., 1987) suggests that an analogous primary processing event may occur in this organism as well, although this has been difficult to determine since the entire 5’ external transcribed spacer of yeast pre-rRNA is extremely short-lived (Veinot-Drebot et al., 1988). The requirement of U3 for yeast viability (Hughes et al., 1987) indicates that one (or more) processing steps in which U3 participates is essential. In addition to U3, yeast possess at least eight other nucleolar snRNA species (Tollervey and Guthrie, 1985; R. Parker et al., 1988; Zagorski et al., 1988), at least two of which, snRl0 (Tollervey and Guthrie, 1985; Tollervey, 1987) and snR128 (Zagorski et al., 1988; Li et al., 1990), appear to play important roles in rRNA processing. While the snR128 species is evidently involved in 18s maturation (Li et al., 1990), deletion of the yeast gene for snR10, which like U3 can associate specifically with the primary rRNA transcript, interferes with the initial processing of the rRNA precursor (Tollervey and Guthrie, 1985; Tollervey, 1987). However, the exact processing sites at which these yeast snRNAs function remain to be defined. The fibrillar regions within nucleoli are the sites of transcription of rRNA. Both RNA polymerase I and topoisom-

erase I were initially localized to these regions by various immunocytochemical techniques (reviewed in Sommerville, 1986; Jordan, 1987). Subsequently, a novel antigen recognized by autoantibodies from scleroderma patients was also found to be localized in the fibrillar centers and dense fibrillar regions of the nucleolus (Ochs et al., 1985); hence the name “fibrillarin.” Our finding that the U3 RNP, which contains fibrillarin, participates in primary rRNA processing is compatible with the antigenic localization of fibrillarin since this processing event occurs on nascent transcripts or soon after the transcripts are completed (Miller and Sollner-Webb, 1981; Gurney, 1985). Electron microscopic spreads of transcribing rRNA genes reveal a gradient of closely spaced nascent RNAs associated with protein that have been dubbed “Christmas trees” (Miller and Beatty, 1969). At the 5’ end of the nascent rRNA fibrils is a distinctive electron-dense particle termed a “terminal knob.” Although the terminal knobs have long been recognized as characteristic of rRNA gene transcription units and are found in widely differing species, neither their biological function nor their composition has been defined. We propose that the terminal knobs are primary rRNA processing complexes. Consistent with this hypothesis are several of our in vitro observations. First, we have shown that the complex containing the U3 snRNP assembles on the primary processing site near the 5’ end of precursor rRNA and remains attached to the downstream rRNA following the cleavage reaction (Figures 4 and 5; S. K. and 8. S., unpublished data). Second, the primary rRNA processing complex is a large structure of at least 20s that forms quickly (S. K. and B. S., unpublished data). Clearly, there are sufficient U3 snRNPs in a nucleolus (an approximately 2- to lo-fold excess relative to the number of pre-rRNA molecules [Prestayko et al., 19701) that such a complex could form very rapidly and then be maintained on each nascent prerRNA in vivo. Notably, the U RNA-containing complexes that form at the intronlexon boundaries of pre-mRNA splicing substrates appear in the electron microscope as similar electron-dense particles (Osheim et al., 1985; Beyer and Osheim, 1988). The strong likelihood that the classical 5’terminal knobs represent the primary rRNA processing complex suggests that this U3 snRNP-dependent reaction is ubiquitous, playing a central role in eukaryotic rRNA maturation. Experimental Procedures rRNA Substrates and Processing Reactions Templates for the T7 RNA polymerase-synthesized rRNA contain mouse rDNA sequences extending between the indicated positions (312, 645, or 669 to 1290, isolated from a 5’ deletion series [Craig et al., 1967)) and cloned between the EcoRl and Pstl sites in pGEM3. The rRNA substrates were synthesized in the presence of [a-32PpP (Kass et al., 1967) and gel purified (Peattie, 1979) before use. Mouse S-100 cell extracts were prepared as desaibed (Miller and Sollner-Webb. 1961) from mid-log phase as&e&grown Ehrlich Ascites cells (Tower and Sollner-Webb. 1967) or from mid-log phase L1210 tissue culture cells. The experiment of Figure 1 used a nuclear extract prepared according to described procedures (Dignam et al., 1983) from actinomycin D-treated Ehrlich Ascites cells (lbwer and SollnerWebb, 1967). The 25 ~1 processing reactions contained 1.5-6 WI of cell extract in a solution containing 120 mM KCI, 20 mM HEPES (pli 7.9),

Cell 906

2 mM MgCiz, 2 mM DTT, 0.14 mM EDTA, 9% (v/v) glycerol, 1.5 mM ATP, 0.0084 pmol of the rRNA substrate, unless otherwise indicated. Reactions were for 45 min at 30°C and RNA was isolated and analyzed on urea-containing 4% polyacryiamide gels as described (Miller and Sollner-Webb, 1981). Mlcrococcal Nuclaasa Digesiion Six microliters of extract was pretreated with 2 pg nuclease at 30% for the times indicated in the figures. tions contained the salts from the extract plus 1 mM EGTA, where indicated), and 2.5 ul was removed and processing reaction, containing a final concentration to inhibit the nuclease.

of micrococcal The 10 ul reacCaClz (or 3 mM used in a 25 ul of 0.3 mM EGTA

RNAase H Dlgastion Oligonucleotides provided by Dr. John Flory (Yale University) were synthesized on an Applied Biosystems DNA Synthesizer and purified by gel electrophoresis. The following oligonucleotides were used: U3 5-20 ATCCCTGAAAGTATAG, U3 17-31 CTATAGAAATGATCC, U3 6479 GTGGTTTCGGGTGCTC, U3 80-96 CGCTACGTCTCGTCCTC, U3 105-120 GCCGGCTTCACGCTCA. U3 122-136 TCAAGCAGCACCTAG, U3 154-168 GAACGATCATCAATG, U3 201-215 TCCACTCAGACTGCG, U118 CAGGTAAGGATT and U&L15 CAGATACTACACTTG. The 15 ul reactions included 10 trl of Ll210 cells S-100 extract, 1 ug of the appropriate oligonucleotide, 2 U of RNAase H (BRL) in a solution containing 206 mM KCI, 4 mM MgClz, 33 mM HEPES (pH 7.9). 18% (v/v) glycerol, 3.3 mM DTT. After 10 min at 3pC and 20 min at 3oOC, reactions were treated with 4 U of DNAase I (Worthington) for 3 min at 3PC followed by 7 min at 30°C. Forty percent of the reaction mixture was removed (for Northern analysis of the remaining snRNAs; see below) and the remainder was brought to 15 ul by addition of 0.004 pmol of 3*P-labeled substrate (rRNA residues 312 to 1290) and 26 nmol of ATP (to achieve a final free MgClz concentration of -0.5 mM). These processing reactions were incubated for 40 min at 30°C, and their RNA was isolated and analyzed on 4% denaturing polyacrylamide gels. Northern Slot Analyses of snRNAs RNAs fractionated in 10% denaturing polyacrylamide gels were electrotransferred onto Zeta-Probe membranes (Bio-Rad) at 4% for 15 hr at 206 mA in 0.5x TBE buffer; then the membranes were dried for 1 hr at 8oOC in a vacuum drier. Hybridization with the U3 and U4 probe was performed according to the membrane suppliers protocol, except that formamide was omitted from the hybridization solutions. After hybridization, blots were washed in 2x SSC, 0.1% SDS then in 0.5x SSC, 0.1% SDS for 30 min, each time at room temperature. The pSP65 plasmid containing a rat U3B RNA insert (Stroke and Weiner, 1985, 1989) was kindly provided by I. Stroke and A. Weiner (Yale University), and the pSP64 plasmid containing U4 RNA insert was provided by D. Black (Black and Pinto, 1989). To generate hybridization probes, the plasmids were linearized with Hindlll and transcribed in vitro with SP6 polymerase in the presence of [@P]UTP Antibodies and lmmunoprecipitatlon The monocional 7289 antibody was obtained from G. Reimer and E. Tan of the Scripps Institute (Reimer et al., 1987) and patient antifibriilarin sera were from G. Reimer (Di) and R. Sontheimer, University of Texas, Dallas (JH). The monoclonal anti-Sm antibody (Y12) was prepared by Mei-Di Shu (Lerner et al., 1981). For immunodepletion, U210 cell S-100 extracts were diluted C-fold in a buffer containing 20 mM HEPES (pH 7.9) 130 mM KCI, 1.25 mM MgCIz, 1.9 mM ATP, 2 mM DTT, 0.125 mM EDTA, 7% glycerol and were incubated for 1 hr at 4OC with antibodies prebound to Protein A-Sepharose as described (Gold et al., 1988). After incubation, the immunoprecipitation mixtures were spun for 10 s in a microfuge, and the supernatants were collected and assayed for the processing activity. The pellets were washed five times in NET-2 buffer (50 mM Tris-HCI [pH 7.51, 150 mM NaCI, 0.05% Nonidet P-40) and RNA was isolated by phenol extraction. For immunoprecipitation of processing complexes, a 20 ui standard processing reaction involving L1210 cell S-100 extract was diluted IOfold in a buffer containing 20 mM HEPES (pH 7.9) 100 mM KCI, 1 mM MgCIz, and the precipitation was performed as described (Gold et al., 1988).

Mobility Shift Gels and Northern Analysis A 12.5 pl processing reaction containing 1.5 ul of Ascites cell S-100 extract and 0.08 pmol of substrate rRNA was incubated for 50 min at 30°C, diluted with an equal volume of processing buffer, and then incubated for an additional 10 min in the presence or absence of 26 ug/ml heparin. Then, 15 ul was electrophoresed on a 2.5 mm thick 4% polyacrylamide gel (651 acrylamide to bis ratio) containing electrophoresis buffer (45 mM Tris-borate [pH 8.31 and 0.45 mM EDTA). The gel was then treated with a solution of 0.5 mg/ml proteinase K (in electrophoresis buffer plus 0.5% SDS) for 45 min at 3pc and electroblotted onto a Gene Screen Plus membrane. The filter was UV crosslinked, baked, prehybridized, and hybridized as described (Konarska and Sharp, 1987). For reprobing, the membrane was stripped using three changes of boiling buffer (Konarska and Sharp, 1987). The esP-sub stituted U3 probe was prepared from the U3B clone (Stroke and Weiner, 1985, 1989) linearized with Pvull, and transcribed with SP6 polymerase; the rRNA probe contained the complement of residues 645 to 1290, transcribed with SP6 polymerase from an EcoRi-cleaved rDNA 645-1290 template. Nuclaoslde Polyphosphata Depletion To deplete extract of endogenous nucleoside polyphosphates, 4 ul of Ascites cell S-100 extract was incubated in a total volume of 50 ul containing processing buffer, 22 U/u1 of bacterial alkaline phosphatase and 10 uCi of (r@P]CTP for 25 min at 3pC. One microliter was spotted onto polyethyleneimine (PEI) paper, which was developed in 0.4 M ammonium sulfate until the buffer front migrated 4 cm and then in 0.7 M ammonium sulfate until the buffer front reached 10 cm. The chromatogram was subjected to autoradiography; unlabeled NTPs, NDPs, and NMPs, run in adjoining tracks as migration standards, were visualized by UV shadowing. Acknowledgments We are indebted to Alan Weiner and members of the Steitz and Sollner-Webb labs for comments on the manuscript. Drs. G. Reimer, E. Tan, and R. Sontheimer generously provided the anti-fibrillarin antibodies and Drs. llana Stroke and Alan Weiner provided the U3 clone. We also thank Nadine Qashu for technical assistance and Lynda Stevens and Margaret Minnek for assistance in preparing the manuscript. This work was supported by ACS grant NP-731A to B. S. and PHF grant GM26154 to J. A. S. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenr” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

15, 1989; revised

January

16, 1990.

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in Proof

Susan Kass and Kasimierz Tyc contributed equally The work referred to as Mougey et al. (unpublished of E. Mougey, L. Pape, and B. Sollner-Webb.

to this work. data) is the work