A possible coordinate role in ribosome biogenesis.

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11471-11476, October 1996 Biochemistry

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: A possible coordinate role in ribosome biogenesis. (endoribonuclease/in situ hybridization/ribonucleoprotein/RNA structure/rRNA)

BENHUR LEE*, A. GREGORY MATERAtt, DAVID C. WARDt§, AND JOE CRAFT*¶ Departments of *Internal Medicine, tGenetics, and §Molecular Biophysics and Biochemistry, Yale University School of Medicine, Box 208031, 333 Cedar Street, New Haven, CT 06520-8031

Communicated by Aaron B. Lerner, Yale University, New Haven, CT, July 18, 1996 (received for review February 23, 1996)

ABSTRACT RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes tRNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-Omethyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in a macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized the RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.

RNP (5, 18), an endoribonuclease that processes precursor tRNAs to generate their mature 5' termini (for review, see ref. 19). The antigenic 40-kDa polypeptide of RNase MRP also binds the RNA component of RNase P (8, 20). In addition to its nuclear function, eukaryotic RNase P has activity in mitochondria (21), although, at least in mammals, its RNA is nuclear encoded (19, 22). The predicted secondary structures of these RNAs reveal that both contain a similar central cage-shaped motif and a conserved pseudoknot, despite their different primary sequences (23, 24). Highly purified preparations of RNase P are also able to aberrantly process a 5.85 pre-rRNA substrate in a similar system used to demonstrate accurate processing of this substrate by RNase MRP (25), a finding confirmed in vivo with mutants in yeast RNase P that are defective in 5.8S rRNA processing, albeit at a different site than RNase MRP mutants (26). The shared polypeptide(s) of RNase MRP and RNase P suggested the possibility that these particles could be physically associated in vivo. The similarities between the secondary structures of their RNAs and the likelihood that they have roles in ribosome biogenesis raised the notion that RNase P could reside in the nucleolus. In this report, we provide biochemical evidence for an association of RNase MRP with RNase P in a large macromolecular complex, possibly preribosomes. We also show that some RNase P RNA resides in the nucleolus, analogous to the RNase MRP RNA. These findings raise the possibility that these two RNA processing enzymes function in a coordinate fashion in ribosome biogenesis.

RNase MRP, a mitochondrial RNA processing enzyme containing a nuclear-encoded RNA, cleaves a primer RNA substrate involved in mtDNA replication in vitro (1, 2). The mammalian RNase MRP RNA is identical to the RNA component of the nucleolar Th ribonucleoprotein (RNP) particle (3-6), which contains several polypeptides in mammalian cells, including an abundant one of 40 kDa (7-9). The identity of the RNA components of these two RNPs suggested that the Th RNP was the nuclear counterpart of RNase MRP, a notion supported by the finding that anti-Th RNP antibodies found in sera of patients with certain autoimmune diseases depleted RNase MRP activity from both nuclear and mitochondrial extracts in vitro (5). Recent ultrastructural evidence has confirmed the presence of the RNase MRP RNA in mitochondria (10); however, the majority of RNase MRP resides in the nucleus (2, 11-13) with localization in the granular region of the nucleolus (4, 7, 14). Genetic deletion experiments in Saccharomyces cerevisiae suggest that the RNase MRP RNA is required for proper maturation of 5.8S rRNA (15, 16). In mammalian cells, nuclear RNase MRP also cosediments with 60-80S particles in vitro, implying an association with ribosomal RNA precursors (17). These facts indicate that RNase MRP plays a role in ribosome biogenesis, in addition to its potential role in mtDNA replication. Autoimmune sera that immunoprecipitate RNase MRP from solution also bring down the ribonuclease P (RNase P)

MATERIALS AND METHODS Oligonucleotide Design and RNase H Digestions. Deoxyoligonucleotides were designed complementary to singlestranded regions of the RNase MRP and RNase P RNAs (Fig. 1A, thin lines). RNase H digestions were performed as described (27). Antisense Affinity Selection. 2'-O-methyl oligoribonucleotides (28) were designed based upon results from RNase H digestion experiments (see Fig. 1 A and B). Biotinylated 2'-O-methyl oligoribonucleotides (Th-7 OMe, Th-12 OMe, Hi-i OMe, and H1-8 OMe) were synthesized using commercially available phosphoramidites, with three biotinylated UTPs successively added onto the 3' end. Antisense affinity selection was carried out as described (29), with purified RNAs 3'-end-labeled with [32P]pCp using T4 RNA ligase (New England Biolabs) (30), and separated in 5% denaturing polyacrylamide gels. Abbreviations: ITS, internal transcribed spacer; MRP, mitochondrial RNA processing enzyme; RNase P, tRNA processing enzyme; RNP, ribonucleoprotein. tPresent address: Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955. ITo whom reprint requests should be addressed.

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. §1734 solely to indicate this fact. 11471

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nick-translated full-length cDNA probe. Imaging and pseudocoloring were as described (31). Glycerol Gradient Fractionation. Log phase HeLa cells were sonicated and cleared by centrifugation (12,000 x g for 30 min). Precleared cell sonicates (100 ,ul) (2 x 107 cells) were layered onto 10-40% linear glycerol gradients containing 100 mM KCl/20 mM Hepes KOH, pH 7.9/5 mm MgCl2/50 ,uM phenylmethylsulfonyl fluoride, followed by centrifugation at 35,000 rpm for 16 hr at 4°C in a SW41Ti rotor (Beckman) and subsequent fractionation into 22 aliquots. U3 and U8 RNAs were used as gradient markers. Northern Blot Analysis. RNA samples from glycerol gradient fractions were resolved on 7% polyacrylamide/7 M urea gels and electroblotted onto Zeta-Probe membranes (BioRad). Probes for RNA blots included SP6 or T7 transcribed RNAs complementary to RNase MRP and RNase P, generated by polymerase chain reaction from plasmids pSpTh and pUCH1, respectively.

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FIG. 1. (A) Secondary structures of the RNase MRP and RNase P RNAs, respectively; adapted from Schmitt et al. (24) as proposed by Forster and Altman (23). The central cage-shaped motif is shown in bold. The conserved pseudoknot is between nucleotides 72 to 78 and 244 to 249 in the RNase MRP RNA and between nucleotides 78 to 83 and 317 to 322 in the RNase P RNA. Oligonucleotides used for RNase H digestions were complementary to the regions indicated by thin lines and 2'-O-methyl oligoribonucleotides were complementary to the regions indicated by bold lines. (B) Oligonucleotide-mediated RNase H digestions of the RNase MRP and RNase P RNAs, using oligodeoxynucleotides shown in A. RNAs were detected by northern blotting. The positions of the intact RNase P RNA (formally labeled as Hi RNA; ref. 33) and RNase MRP RNA are indicated.

In Situ Hybridization. HeLa monolayer cells were prepared and hybridized with antisense oligonucleotides as described (31). The human RNase P RNA was also visualized using a

Oligonucleotide Design for Identification of RNase MRP and RNase P. For oligonucleotide design, we relied upon the predicted secondary structure for the RNase MRP and RNase P RNAs (23, 24, 32). Single-stranded regions were selected and their accessibility within intact RNP monoparticles confirmed by oligodeoxynucleotide-directed RNase H digestions. For example, note that Hi-i and Hl-8 mediated almost complete digestion of RNase P RNA, whereas Th2 mediated >50%, and Th7 nearly complete, digestion of RNase MRP RNA (Fig. 1B). In multiple experiments, Th2, Th 4, Th7, ThlO, and Thll mediated RNase H digestion of RNase MRP RNA, whereas Thl and Th5 did not. Concurrently, H1-1, Hl-8, and Hl-9 mediated digestion of the RNase P RNA, whereas Hi-3, Hi-5, and H1-7 did not. Based on these results, and upon the exclusion of homologous regions between these two RNAs, two antisense 2'-O-methyl RNA oligonucleotides specific for RNase MRP (Th-7 OMe and Th-12 OMe; Fig. 1A Upper) and two specific for RNase P (Hi-i OMe and Hi-8 OMe; Fig. 1A Lower) were constructed for use in affinity selection and in fluorescence in situ hybridization experiments. Th-12 OMe was almost identical to Th2 and overlapped Th7, both showed that the long single-stranded RNA region in RNase MRP is accessible. Affinity Selection of RNase MRP and/or RNase P. To demonstrate an association between RNase MRP and RNase P, biotinylated 2'-O-methyl RNA oligonucleotides were used with streptavidin-conjugated agarose beads to select the individual RNP particles from HeLa extracts (27-29). In these experiments, [32P]pCp 3'-end-labeling of RNAs obtained after antisense affinity selection showed that the RNase MRPspecific oligonucleotides, Th-7 OMe and Th-12 OMe, and RNase P-specific oligonucleotides, Hi-8 OMe (and to a lesser extent, Hi-i OMe), selected their cognate RNAs from equivalent amounts of cell extracts (Fig. 2A, lanes 2-5). The efficiencies of each oligonucleotide differed in that Hi-8 OMe only selected about 20% of Hi, whereas Thl2 OMe and Th7 OMe both selected about 80% of RNase MRP RNA. In multiple experiments, untargeted RNase P RNA also was always coselecfed when using RNase MRP-specific oligoribonucleotides (Fig. 2A, lanes 4 and 5). In contrast, two control 2'-O-methyl RNA oligonucleotides with specificity for the U2 and U6 RNAs, respectively (BU2b and BU6c) did not select either the RNase P or RNase MRP RNAs, although they did bind the U2 and U4/U6 RNAs as expected (Fig. 2A, lanes 6 and 7). In multiple experiments, BU6c always selected U4 and U6 RNAs in roughly equimolar amounts as would be expected from their in vivo relationship (34), although the U6 RNA, along with other low molecular weight -RNAs, including 5S RNA and tRNAs, often migrated off low percentage gels. The


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FIG. 2. (A) Antisense affinity selection with 2'-O-methyl oligoribonucleotides specific for the RNase MRP and RNase P RNAs using whole HeLa extracts. RNA selection was carried out using the antisense biotinylated 2'-O-methyl oligoribonucleotides indicated in Fig. 1, followed by 3' end labeling of precipitated RNA. Equivalent amounts of RNA were loaded onto each lane; however, lane 1 was exposed for one-fifth the time. Lane 1, total HeLa RNA extract; lanes 2-8, antisense affinity selected RNAs, using oligonucleotides specific for the RNase MRP, RNase P, U2 (BU2b), and U6 (BU6c) RNAs; lanes 9-11, immunoprecipitated RNAs using known reference sera. (B) Antisense affinity selection using in vitro transcribed RNase MRP and RNase P (Hi) RNAs. Equimolar amounts from the same transcription reaction, each for RNase MRP RNA and RNase P RNA, were loaded into each lane so that selection efficiencies could be compared.

lack of RNase P RNA in these control lanes, and in the lane containing RNAs selected with streptavidin-conjugated agarose beads (Fig. 2A, lane 8, no oligonucleotide control), indicated that the selection of the RNase P RNA by RNase MRP antisense oligomers was not due to non-specific inter-

actions. The significant amount of U2 selected by Th-7 OMe (Fig. 2A, lane 4) is likely due to the complementarity between the oligomer and nucleotides 133 and 142 of the U2 RNA. Although the fraction of cognate RNAs selected by the RNase MRP and RNase P specific oligomers did not match the preponderance of that selected by the U2 and U6 specific oligomers, it is important to note it was still a significant fraction when compared with the appropriate controls. For example, note that significant amounts of RNase MRP and RNase P RNA were selected with Th-12 OMe, although the amount of U2, Ul and U4 RNAs selected by this oligomer was approximately equivalent to the background selected by plain streptavidin-agarose beads (Fig. 2A, compare lanes 5 and 8). The specificity of this selection was further supported by the observation that the amounts of Ul and U2 RNAs brought down by Th-12 OMe and BU6c were the same. In addition, with shorter gel running times, Th-7 OMe, Th-12 OMe, BU2b, BU6c, and beads alone always brought down 5S RNA in equivalent amounts (data not shown). In contrast to selection of the RNase P RNA by the RNase MRP RNA-specific 2'-O-methyl oligoribonucleotides, the RNase P specific oligoribonucleotides did not consistently bind the latter RNA (Fig. 2A, lanes 2 and 3). Only in two of five experiments did RNase MRP appear to be selected by a RNase P-specific oligomer, H1-8 OMe (data not shown), and Hi-i OMe never coselected RNase MRP RNA. This inconsistent binding suggests that RNase P is more loosely associated than RNase MRP with higher order complexes in the nucleolus (see below) or that annealing of the 2'-O-methyl oligomers with the RNase P RNA disrupts its interaction with RNase MRP. To ensure that the coselection of RNase P RNA with RNase MRP RNA was not due to cross-hybridization, each 2'-Omethyl oligoribonucleotide was tested for its ability to select in vitro transcribed RNase MRP or RNase P RNA (Fig. 2B). Each oligonucleotide was highly specific for its cognate RNA, although their selection efficiencies varied; neither were selected by a non-cognate oligomer, HY3.1B, specific for one of the Ro RNAs. Thus, our antisense affinity selection experiments suggested that small subpopulations of RNase MRP and RNase P are specifically associated with each other, or are associated via a common macromolecular complex. Glycerol Gradient Fractionations. To explore the above possibilities, whole HeLa extracts were fractionated in glycerol gradients, and RNase MRP and RNase P were detected by probing for their RNA components in Northern blots. Both RNP monoparticles sedimented at roughly 12-15S as previously demonstrated (2, 33), although. in multiple gradients, RNase MRP was always found in slightly lighter fractions than

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FIG. 4. Fluorescence in situ hybridization with RNase MRP and RNase P probes miHeLa monolayer cells. B and E show cells that were hybridized with the Th-12 OMe, H1-8 OMe oligomers, respectively. The cell in K was hybridized with a biotinylated human RNase P cDNA. A, D, and J show

Biochemistry: Lee et al. RNase P (Fig. 3, fractions 8-14 for RNase P [Hi RNA] and fractions 10-16 for RNase MRP [MRP RNA]). In addition to these monoparticles, RNase MRP also cosedimented with heavier structures of about 60-80S (Fig. 3; MRP RNA, fractions 2 and 3) (17). Moreover, a small subpopulation of RNase P cofractionated at the same S values as the macromolecular-complexed RNase MRP (Fig. 3; Hi RNA, fractions 2 and 3). While the potential exists that the sedimentation of RNase P in the 60-80S region was nonspecific, and related to possible ribosomes in cell extracts, Ul RNP, as a control, did not sediment at these S values (data not shown). Overall, these data suggest that the association between small subpopulations of RNase MRP and RNase P demonstrated in the antisense selection experiments may occur within these macromolecular complexes. Initial phosphor image analyses indicates that less than 5% of the total RNase P and RNase MRP are associated in these complexes. Given the location of RNase MRP in the nucleolus (7) and its role in 5.8S rRNA maturation (15, 16), these larger macromolecular complexes are likely ribosomal precursors or preribosomes (35). This notion is corroborated by the finding that a subpopulation of the U3 RNP, previously shown (36) to cosediment with higher ordered structures in the nucleolus of about 70S, cosediments in the same fractions as macromolecular-complexed RNase MRP and RNase P (Fig. 3; U3 RNA, fractions 2 and 3). Intracellular Location of RNase MRP and RNase P. We next determined the intracellular location of the RNA components of these RNPs using our 2'-O-methyl RNA oligonucleotides for in situ hybridization experiments. Anti-RNase MRP RNA oligonucleotides (Fig. 4 B, Th-12 OMe, and H, Th-7 OMe) hybridized to the nucleolus and, to a lesser extent, the cytoplasm, consistent with the role of this RNP in prerRNA processing and mitochondrial DNA replication. Conversely, the anti-RNase P RNA oligonucleotide, H1-8 OMe, hybridized mainly to the cytoplasm, and to a lesser extent, the nucleoplasm and nucleolus (Fig. 4 E and F). The fluorescence patterns seen with the anti-RNase P and anti-RNase MRP RNA probes were not likely due to cross hybridization with rRNA for several reasons. First, database searches failed to identify regions of complementarity with rRNA sequences greater than five or six nucleotides. Second, antisense selection experiments using these same oligoribonucleotides failed to select any rRNAs above the background of the no oligonucleotide control (Fig. 2A). Third, in gylcerol gradient fractionation experiments, northern blotting with antisense DNA oligomers identical in sequence to the RNA 2'-O-methyls used failed to detect any other RNA in total cell extracts (not shown). Fourth, the fluorescence intensities of the anti-RNase MRP and anti-RNase P RNA oligomers were substantially lower than those of an anti-28S control oligonucleotide or anti-rRNA antibodies (not shown). As a further control, we used a nick-translated cDNA as a probe on postextracted HeLa cells (Fig. 4 J-L). Although the fluorescence signal was somewhat weaker than that seen with the anti-H1-8 oligomer, the overall pattern of hybridization was the same: relatively strong in the cytoplasm and the perinucleolar compartments (see ref. 37 and below), with background fluorescence in the nucleoplasm. Human autoantibodies directed against the 40-kDa polypeptide common to both RNase P and RNase MRP stained cells in a manner similar to the antisense oligomers (Fig. 4G), including nucleolar colocalization as depicted by orange coloring of merged images (Fig. 41). Other than the slight nucleolar fluorescence seen in both the autoantibody and the


11475

antisense oligomer patterns, there were two notable differences. First, the relative fluorescence intensity of the antibody staining was greater than that of the oligomers in both the nucleoplasm and the cytoplasm (i.e., the ratio of staining intensity of the nucleoplasm to that of the nucleolus is greater in Fig. 4G than in 4H). The incomplete overlap observed between the antisera and aiiti-RNase MRP oligoribonucleotides may be accounted for by pools of RNase MRP proteins, identified in sucrose density gradient fractions, that are not assembled with their cognate RNA (data not shown). Second, a bright dot of fluorescence was detected at the periphery of one or more nucleoli per cell when the antisense probes were used, a finding not seen with the antiserum (compare Fig. 4 B, E, and H with 4G). This dot sometimes had a track-like appearance. These novel structures are called perinucleolar compartments and have been suggested to be sites for accumulation of certain RNA polymerase III transcripts using some of the antisense oligomers presented herein (37).

DISCUSSION In Vivo Association of RNase MRP and RNase P. Our experiments have shown that small subpopulations of RNase MRP and RNase P are associated, and that this association may be in the nucleolus, as determined by in situ hybridization with specific probes. However, our antisense selection experiments and the glycerol gradient fractionations are unable to determine if this association is a direct one between the two particles, or an indirect one via a macromolecular complex such as preribosomes. Nevertheless, these results are consistent with the notion that RNase P may function in rRNA processing, a hypothesis raised by Clayton (38), and compatible with the role of RNase MRP in the nucleolus. Possible involvement of RNase P in Ribosomal Biogenesis. In S. cerevisiae, RNase MRP apparently cleaves a site near the 3'-end of the internal transcribed spacer (ITS1) in pre-rRNA (15, 16), a notion supported by recent in vitro data demonstrating processing at this site by the purified enzyme (25). It is notable that the intervening sequence between the 16S and 23S rRNA in prokaryotes contains spacer tRNAs, efficiently cleaved by RNases P and D in subsequent processing steps (39, 40). As a postulate, if ITS1 in eukaryotes is considered as the analogous region occupied by the spacer tRNAs in prokaryotes, then conservation in secondary or tertiary structure

could make certain regions of ITS1 susceptible to cleavage by RNase P (Fig. 5). Indeed, the significant divergence in primary structure across species seems to have little effect on the secondary structure adopted by the ITS1, and recent findings suggest the possibility that purified RNase P may cleave this substrate in vitro (25). Three stem-loop motifs have apparently been conserved from yeast to humans (41), suggesting that such conservation may be relevant to transcript processing. Although this analysis has not been extended to prokaryotes, it stands to reason that RNase P and RNase MRP may have evolutionary relatedness selected by action on such an ancestral substrate (42). The structural and functional similarities of these two enzymes are underscored by the finding that RNase P can cleave at site 2 of the mouse D-loop mtRNA, the canonical substrate for RNase MRP (43). Further structural analysis of this RNA and of ITS1 and pre-5.8S rRNA will be required to discern if they bear any secondary or tertiary structural motifs that may be similar to those of pre-tRNAs. Cytoplasmic Presence of RNase MRP and RNase P. Our in situ hybridization experiments indicate that the RNase MRP

the nuclei within the same fields, stained with 4',6-diamidino-2-phenylindole. G represents cells stained with anti-Th autoantibodies (5) and H shows hybridization with another RNase MRP specific oligoprobe, Th-7 OMe. C, F, I, and L are pseudocolored and merged versions of the black and white source images shown to the left of each panel. Note the bright dots of fluorescence near the periphery of some of the nucleoli (B, E, H, and K) that were not detected by the anti-28S probe (not shown). These so-called perinucleolar compartments (37) are sometimes found to occupy small cavities at the nucleolar border (arrows).

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Proc. Natl. Acad. Sci. USA 93 (1996) probes, and Sandra Wolin for the HY3.1B oligomer; all are at the Yale University School of Medicine. We also thank David Clayton and Sidney Altman for plasmids pSpTh and pUCH1, respectively. This work was supported in part by grants from the National Institutes of Health (AR 42475 and AR 40072) to J.C. B.L. was supported by a Research Training Fellowship for Medical Student's from the Howard Hughes Medical Institute, and A.G.M. was funded in part by fellowship DRG-1135 from the Damon Runyon-Walter Winchell Cancer Research Fund.

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and RNase P RNAs are localized to the cytoplasm, in addition to the nucleolus. The finding that cytoplasmic fluorescence for the RNase MRP RNA is equal to, or less than, the combined nucleolar fluorescence can be reconciled with recent ultrastructural data that showed the distribution of gold particles after in situ hybridization with biotinylated antisense RNA probes was approximately equivalent per Aum2 over the nucleolar and mitochondrial compartments (10). The cytoplasmic fluorescence seen in our in situ hybridizations may represent mtRNase P RNAs; however, in contrast to RNase MRP, a majority of the antisense hybridization signal from the RNase P RNA was detected in the cytoplasm. In interpreting this result, it is important to consider that in biochemical purification of HeLa RNase P, a significant amount of the enzyme activity is found in the cytosolic fraction (21, 33), and in purification of mtRNase P, the majority of the enzyme activity is located in the postnuclear, postmitochondrial fraction (21). Even so, it is not clear that nuclear leakage of RNase P can account for all of the cytoplasmic fluorescence, raising the question of an intrinsic presence of RNase P in the cytoplasm. Multiple functions have been established for RNase P in prokaryotes; in addition to processing of pretRNAs, RNase P is also involved in the processing of 4.5S RNA in Escherichia coli (44), the homolog of mammalian SRP RNA (reviewed in 45), and polycistronic mRNAs (46). If multiple roles for RNase P are preserved in eukaryotes, some may take place in the cytoplasm. RNase MRP and RNase P: Dynamic Roles in Cellular Regulation? The association of RNase MRP with RNase P in the nucleolus may have functional consequences for the cellular regulation of protein synthesis. Coupling tRNA synthesis and maturation with ribosomal biogenesis by controlling the levels of RNase MRP and RNase P may offer a way to optimize ratios between the components of cellular protein synthesizing machinery. The RNA components of both these RNPs are RNA polymerase III transcripts with highly conserved 5' flanking regions and almost identical type 3 promoter structures (11, 22), and given the similarities in their 5'-flanking and intragenic regions (they both have a nonfunctional Box A in the 3'-half of their coding regions), it is not unreasonable to suppose that these two RNAs may be responsive to the same transcriptional signals. We thank Susan Baserga for helpful comments, Joan Steitz for providing the antisense 2'-0-methyl oligoribonucleotides specific for the U2 and U6 RNAs and the plasmids for generating the U3 and U8

Chang, D. D. & Clayton, D. A. (1987) Science 235, 1178-1184. Chang, D. D. & Clayton, D. A. (1987) EMBO J. 6, 409-417. Hashimoto, C. & Steitz, J. A. (1983) J. Biol. Chem. 258, 1379-1382. Reddy, R., Tan, E. M., Henning, D., Nohga, K. & Busch, H. (1983) J. Biol. Chem. 258, 1383-1386. 5. Gold, H. A., Topper, J. W., Clayton, D. A. & Craft, J. (1989) Science 245, 1377-1380. 6. Yuan, Y., Singh, R. & Reddy, R. (1989) J. Biol. Chem. 264, 14835-14839. 7. Reimer, G., Raska, I., Scheer, U. & Tan, E. M. (1988) Exp. Cell Res. 176,

1. 2. 3. 4.

117-128. 8. Yuan, Y., Tan, E. M. & Reddy, R. (1991) Mol. Cell. Biol. 11, 5266-5274. 9. Kipnis, R. J., Craft, J. & Hardin, J. A. (1990) Arthritis Rheum. 33, 14311437. 10. Li, K., Smagula, C. S., Parsons, W. J., Richardson, J. A., Gonzalez, M., Hagler, H. K. & Williams, R. S. (1994) J. Cell Biol. 124, 871-882. 11. Topper, J. N. & Clayton, D. A. (1990) Nucleic Acids Res. 18, 793-799. 12. Karwan, R., Bennett, J. L. & Clayton, D. A. (1991) Genes Dev. 5, 12641276. 13. Kiss, T. & Filipowicz, W. (1992) Cell 70, 11-20. 14. Reddy, R., Li, W. Y., Henning, D., Choi, Y. C., Nohga, K. & Busch, H. (1981) J. Biol. Chem. 256, 8452-8457. 15. Schmitt, M. E. & Clayton, D. A. (1993) Mo. Cell. Biol. 13, 7935-7941. 16. Chu, S., Archer, R. H., Zengel, J. M. & Lindahl, L. (1994) Proc. Natl. Acad. Sci. USA 91, 659-663. 17. Kiss, T., Marshallsay, C. & Filipowicz, W. (1992) EMBO J. 11, 3737-3746. 18. Gold, H. A, Craft, J., Hardin, J. A., Bartkiewicz, M. & Altman, S. (1988) Proc. Natl. Acad. Sci. USA 85, 5483-5487. 19. Altman, S, Kirsebom, L. & Talbot, S. (1993) FASEB J. 7, 7-14. 20. Liu, M.-H., Yuan, Y. & Reddy, R. (1994) Mol. Cell. Biochem. 130, 75-82. 21. Doersen, C. J., Guerrier-Takada, C., Altman, S. & Attardi, G. (1985) J. Biol. Chem. 260, 5942-5949. 22. Baer, M., Nilsen, T. W., Costigan, C. & Altman, S. (1989) NucleicAcids Res. 18, 97-103. 23. Forster, A. C. & Altman, S. (1990) Cell 62, 407-409. 24. Schmitt, M. E., Bennett, J. L., Dairaghi, D. J. & Clayton, D. A. (1993) FASEB J. 7, 208-213. 25. Lygerou, Z., Allmang, C., Tollervy, D. & Seraphin, B. (1996) Science 272, 268-270. 26. Chamberlain, J. R., Pagan-Ramos, E., Kindelberger, D. W. & Engelke, D. R. (1996) Nucleic Acids Res. 24, 3158-3166. 27. Blencowe, B. J., Sproat, B. S., Ryder, U., Barabino, S. & Lamond, A. I. (1989) Cell 59, 531-539. 28. Sproat, B. S., Lamond, A. I., Beijer, R., Neuner, P. & Ryder, U. (1989) Nucleic Acids Res. 17, 3373-3386. 29. Wasserman, D. A. & Steitz, J. A. (1991) Mol. Cell. Biol. 11, 3432-3445. 30. England, T. E. & Uhlenbeck, 0. C. (1978) Nature (London) 275, 560-561. 31. Matera, A. G. & Ward, D. C. (1993) J. Cell Biol. 121, 715-727. 32. Topper, J. N. & Clayton, D. A. (1990) J. Biol. Chem. 265, 13254-13262. 33. Bartkiewicz, M., Gold, H. & Altman, S. (1989) Genes Dev. 3, 488-499. 34. Hashimoto, C. & Steitz, J. A. (1984) Nucleic Acids Res. 12, 3283-3293. 35. Hadjiolov, A. A. (1985) The Nucleolus and Ribosome Biogenesis (Springer,

Berlin). 36. Tyc, K. & Steitz, J. (1989) EMBO J. 8, 3113-3119. 37. Matera, A. G., Frey, M. R., Margelot, K. & Wolin, S. L. (1995) J. Cell Biol. 129, 1181-1193. 38. Clayton, D. A. (1994) Proc. Natl. Acad. Sci. USA 91, 4615-4616. 39. Srivastava, A. K. & Schlessinger, D. (1990) Annu. Rev. Microbiol. 44, 105-129. 40. Apirion, D. & Miczak, A. (1993) BioEssays 15, 113-120. 41. Yeh, L. C., Thweatt, R. & Lee, J. C. (1990) Biochemistry 29, 5911-5918. 42. Morrisey, J. P. & Tollervey, D. (1995) Trends Biol. Sci. 20, 78-82. 43. Potuschak, T., Rossmanith, W. & Karwan, R. (1993) Nucleic Acids Res. 21, 3239-3243. 44. Bothwell, A. L. M., Garber, R. & Altman, S. (1976) J. Biol. Chem. 251, 7709-7716. 45. Wolin, S. L. (1994) Cell 77, 787-790. 46. Alifano, P., Rivellini, F., Piscitelli, C., Arraiano, C. M., Bruni, C. & Carlomagno, M. S. (1994) Genes Dev. 15, 3021-3031. 47. Pace, N. R. & Burgin, A. B. (1990) in The Ribosome, ed. Hill, W. E. (Am. Soc. Microbiol., Washington, DC), p. 418.