Conserved Terminal Hairpin Sequences of Histone Mrna Precursors Are Not Involved in Duplex Formation with the U7 RNA but Act As

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4345-4349, June 1989 Biochemistry Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor (histone gene expression/RNA processing/U7 small nuclear ribonucleoprotein particle/anti-Sm antibodies/sea urchin embryos) ALAIN P. VASSEROT*, FREDERICK J. SCHAUFELEt*, AND MAX L. BIRNSTIEL* *Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria; and tInstitut fur Molekularbiologie II der Universitat Zurich, Hcnggerberg HPM-2, CH-8093 Zurich, Switzerland Contributed by Max L. Birnstiel, February 16, 1989

ABSTRACT The hairpin loop structure and the down- A second class of stem-loop structures comprises those stream spacer element of histone mRNA precursors are both hairpins that have in common a requirement for specific needed for efficient 3' end formation in vivo and in vitro. sequences. In this case, the loop sequences and, sometimes, Though generally considered as a single processing signal, these protruding unpaired nucleotides in the stem are a feature two motifs are involved in different types ofinteraction with the essential for function. Here, the primary sequence ofthe stem processing machinery. Whereas RNA duplex formation be- does not seem to be required for recognition, as exemplified tween the downstream spacer element and the U7 small nuclear by the interaction between the R17 coat protein and its RNA is essential for processing, we show here that base pairing palindromic binding site (19). Although the protein-RNA between the histone stem-oop structure and the U7 RNA is not interaction has not been directly investigated, a similar relevant. Our experiments demonstrate that a processing fac- situation probably holds for the binding site of the Li tor other than the U7 RNA makes contact with the highly ribosomal protein (20), for the iron-responsive element of conserved hairpin structure of the histone precursor. The ferritin and transferrin receptor mRNAs (11), and for the recognition of the target site by the processing factor is sequence within the human immunodeficiency virus tar site structure and sequence specific. Prevention of this interaction (21). results in an 80% of3' We have undertaken a detailed analysis of the highly decrease cleavage efficiency in vitro. The conserved terminal stem-loop structure of histone mRNA hairpin binding factor is Sm-precipitable and can be partially precursors (pre-mRNA). We show that in contrast to the separated from the U7 small nuclear ribonucleoprotein particle hairpins above, the histone stem-loop must retain not only its on a Mono Q column. structure but also its overall primary sequence for efficient 3' processing. We further suggest that the hairpin structure is There are many cases where RNA hairpins mediate control not involved in Watson-Crick type base pairing with the U7 ofgene expression. In prokaryotes, stem-loop structures are snRNA, as considered possible (22), but makes contact to involved in attenuation (1), transcription termination (1), another processing factor. This factor is Sm-precipitable and, rRNA processing (2), mRNA turnover (3), retroregulation upon fractionation of nuclear extract, is present in a fraction (3), and translational regulation (4). Their role in ribosome lacking the U7 snRNP and the heat-labile factor previously assembly has also been demonstrated (5). shown to promote 3' processing of histone pre-mRNAs (for In eukaryotes, hairpin structures have been shown to review, see refs. 6 and 7). mediate 3' end formation (6, 7), the regulation (8) or stability (9) of histone mRNAs, the iron response of ferritin (10) and transferrin receptor (11) biosynthesis, as well as modulation MATERIALS AND METHODS of translation (12). A possible role of a folded structure at the Multistep Mutagenesis, Microi'Jection, and Mutant Analy- trans-esterification active site in self-splicing (13), in recog- sis. H3 histone mutants were generated by BAL-31 deletion nition events (14), or in determining the amount ofalternative from a restriction site on the 3' side of the hairpin structure splicing (15) has been suggested. Finally, reports have em- to the first dinucleotide of the stem (GG) resulting in the phasized the role of stem-loop structures in small nuclear formation ofa "left arm" ofthe H3 gene. A similar procedure ribonucleoprotein particle (snRNP) assembly (16, 17). resulted in the formation of a "right arm" starting with the In some cases, the hairpin structure, rather than the last stem dinucleotide (CC). Ligating oligonucleotides in primary sequenceperse, seems to be ofmajor importance for between the left and right arms of the H3 gene resulted in the function. In bacterial attenuation, for instance, synthetic mutants depicted in Fig. 2A. Sea urchin microinjections (23) terminator hairpins are able to promote transcription termi- and RNA analysis were carried out essentially as described nation irrespective of their primary sequence (18). In general (24). increasing stability of the stem-loop structure correlates In Vitro Processing, Processing Substrates, and Competitor positively with the efficiency of transcription termination (1) RNAs. Preparation of nuclear extracts (25), SP6 transcription although some mutations do not follow this rule. The primary (26), in vitro pre-mRNA processing (27), and depletion of sequence of the sea urchin U7 small nuclear RNA (snRNA) Sm-type snRNPs in nuclear extract (28) were performed as hairpin, but not its structure, can be extensively modified described (27, 29). T7 transcription was performed under SP6 without affecting the assembly into an Sm-precipitable par- buffer conditions. To test for complementation, a 10-fold ticle or the processing function of the reconstituted U7 molar excess of a construct with a wild-type palindromic snRNP (17). Abbreviations: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein particle; pre-mRNA, mRNA precursor; HBF, The publication costs of this article were defrayed in part by page charge hairpin binding factor. payment. This article must therefore be hereby marked "advertisement" tPresent address: Metabolic Research Unit, 1141-HSW, University in accordance with 18 U.S.C. §1734 solely to indicate this fact. of California, San Francisco, CA 94143. 4345 Downloaded by guest on September 27, 2021 4346 Biochemistry: Vasserot et al. Proc. Natl. Acad. Sci. USA 96 (1989)

sequence (wtpal compRNA; see text) was added to the \ reaction. Wild-type and mutant pre-mRNA hairpin se- A fL =T 0 quences are shown in Fig. 3A. Oligonucleotides were ligated into a HindIII/Pst I-digested pSP64 (processing substrates) or pSPT19 vector (competitor RNAs). The processing substrates have the following overall sequence: 5'-m7GpppGA- AUACAAGCUUUCCCUAAC-(hairpin)-AACCACAGUC- H3Ba1 H3Baf2 H3Ba10 H3wt UCUUCAGGAGAGCUGACACUGAC-3'. The deltapal and Cpal constructs (see Fig. 3A) have been described elsewhere (30). The competitor RNAs have the following overall sequence: 5'-GGGAGACCCAAGCUUCCCUAAC-(hairpin)- D B H3 3 AACCACAGUCUCUUC-3'. The falsepal compRNA (see To~~ +0-*read text) whose hairpin sequence is 5'-CUGAGAGAAAUCU- 0 through CAG-3' is not shown in Fig. 3A.

RESULTS Duw~e .4- H3 3' 3' Processing in Sea Urchin Embryos Injected with Cloned Histone Genes. In early experiments, it was shown that deletion of the terminal hairpin of sea urchin H2A precursors pow 14H2B 3' results in a processing deficiency in the frog oocyte (31). A possible interpretation was that hairpin deletion prevents FIG. 2. In vivo processing of Psammechinus miliaris H3 wild- essential RNARNA contacts in the hypothetical processing type and mutant pre-mRNAs. (A) Hairpin loop sequences. Mutations complex between the U7 snRNA and the histone pre-mRNA are shown white on black and potential base complementarities with (ref. 22 and Fig. 1). We investigated this hypothesis by the U7 snRNA are marked with an open circle. (B) SP6 mapping. introducing base changes into the stem-loop structure of the H2B 3', 3' end mapping of H2B transcripts as a positive injection sea urchin Psammechinus miliaris H3 early histone gene in control; H3 3', properly processed H3 transcripts; H3 3' read- such a way that the capacity ofthe mutant transcripts to base through, unprocessed H3 transcripts; P, undigested H3 probe; M, pair with the U7 snRNA was severely reduced (Fig. 2A, Hpa II-digested pBR322 marker. potential base pairings are marked with an open circle). The mutants were tested in vivo by microinjection into sea urchin transcripts from a coinjected H2B gene. Fig. 2B shows that eggs of a closely related species, Paracentrotus lividus (23). processed RNA was obtained from both wild-type and all At early blastula, total RNA was extracted, the 3' end of the mutant H3 genes, although the ratios between the mature H3 different mutants was analyzed by SP6 mapping, and the and H2B mRNA pools vary. Interestingly, the H3Ba2 mu- injection efficiency was standardized by comparison to the tant, in which 11 out of 13 possible complementary bases were exchanged (while maintaining a hairpin structure) still HISTONE GA(U)A yielded processed RNA in appreciable amounts. PRE -mRNA U HISTONE 5' It can be concluded from these experiments that produc- PRE - mRNA U7 RNA tion of mature histone H3 mRNA is not critically dependent 3N CAAU U7RNA on the base-pairing capacity between the histone hairpin and N the U7 snRNA, although it has been shown (24) to be dependent on duplex formation between the downstream histone spacer element and the 5' terminal sequences of the U7 snRNA. CC C It should be noted that for all of the mutants, but not the A C U u UU wild-type gene, unprocessed RNA molecules appeared. This, U combined with a somewhat reduced level of mature H3 G G A mRNA by comparison to the H2B standard, suggests that CA U C U processing is affected for the mutants but to an extent that is difficult to quantify precisely because the incubation time required to obtain mappable transcripts is relatively long and the injected developing embryos are a dynamic system ex- hibiting important RNA turnover (ref. 33 and references therein). We, therefore, decided to extend the above obser- I G *C vations by studying 3' processing of mutant transcripts in a U -A - GUGCCCMC(U)-3' /A C *G homologous in vitro processing system where RNA turnover k G-CCGC ,C *G, and RNA degradation are not occurring (27), so that the AGU GU amounts ofprocessed RNA should be a bona fide measure 'U A' of C*G A G the actual processing rate, allowing us to comprehend the C*G A A ,3 finer G*C ,,/ A U details of the reaction. AUb A C 3' Processing in Vitro. SP6 polymerase-generated tran- 'A-G' AC& scripts from H4 mouse histone constructs with various mu- SEA URCHIN MOUSE tations or a deletion ofthe 3' region (Fig. 3A) were processed FIG. 1. Base complementarity between the 3' region of the in vitro in a nuclear extract from mouse hybridoma cells as histone pre-mRNA and the U7 snRNA. The hypothetical RNA described (27). The reaction products were analyzed on duplex between the histone hairpin structure and the U7 snRNA has polyacrylamide gels and the processing efficiency was quan- an estimated free energy (32) of -11 kcal in sea urchin and -7 kcal tified. Depending on the experiment, 50-60% ofthe wild-type in mouse (1 cal = 4.184 J). The two histone conserved elements are input RNA was processed (Fig. 3B, lane 2). This amount was boxed and the hairpin loop is marked by inverted arrows. The mature then taken as 100% for comparison with other processing 3' end of the histone mRNA is designated by an arrowhead. reactions. Downloaded by guest on September 27, 2021 Biochemistry: Vasserot et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4347 A sequences is not absolute because hairpin revertants (see figure 1 of ref. 35) with four stem nucleotides altered yield 0 .. mature RNA in the frog oocyte system (35). All the processed mutant RNAs migrate differently relative IC G1 0 (Cc1011 to the processed wild-type RNA. However, it was shown by 'lGc*?JG.0I~ nc.oGC the pCp-tagging approach (27) that all mutants produce .0Ce~ ~~~a mature RNA species with genuine 3' ends (A.P.V. and H4wt fstemlwtloop false pal open pal delta pal C pal M.L.B., unpublished results). Similar aberrant migration of RNAs has also been reported and results probably from differential destabilization of RNA secondary structure during electrophoresis (36). B 3 a Competition Experiments. Since all alterations of the histone hairpin lead to some reduction of processing, it is not _so INPUT possible to rule out from the above experiments that base complementarity may play a minor role in 3' processing. However, the hairpin nucleotides could interact with a pro- * m ] PROC. cessing factor by mechanisms that do not involve RNA-RNA duplex formation. The strict conservation of the stem-loop INPUT [ -_ nucleotides (34) suggests that such an interaction would be extremely specific. Thus, it should be possible to prevent this interaction by adding a suitable unlabeled competitor RNA and, hence, to inhibit processing of the labeled reporter substrate. To trap a putative hairpin binding factor(s) PROCI[ [HBF(s)], competitor RNAs with various hairpin sequences were used that are themselves not capable ofbeing processed since they lack the downstream spacer element (30). When the competition is carried out with an RNA con- 1 2 3 4 5 6 7 89 taining the wild-type palindrome sequence (referred to as wtpal compRNA), FIG. 3. In vitro processing of mouse H4 wild-type and mutant processing of the labeled wild-type sub- pre-mRNAs. (A) Hairpin loop sequences. Mutations are shown white strate is reduced, with competition leveling off at about 20% on black, potential base complementarities with the U7 snRNA are of the value obtained in the absence of competitor (Fig. 4 B marked with an open circle. (B) In vitro processing. Lanes: 1, and C). Some processing component accounting for 80%o of wild-type pre-mRNA input; 2, 4-7, and 9, processing efficiency of the processing rate is apparently being preempted by the the various mutants and their controls; 3 and 8, Hpa II-digested addition of the wtpal compRNA. That this competition is pBR322. Proc., processed RNA. sequence specific and pertains to the hairpin structure can be concluded from the inability of a competitor RNA with a We first investigated the importance of the highly con- different palindrome sequence (i.e., falsepal compRNA; see served hairpin nucleotides (34). Providing a template with a Fig. 4C) to reduce processing. false palindrome (referred to as the falsepal mutant, see Fig. The downstream spacer element alone is a competitor at 3A) that maintains only two potential contact sites with the high input (Fig. 4C) whereas the complete wild-type RNA U7 snRNA allows processing of the precursor at 17-22% of (having both the conserved stem-oop and the downstream the wild-type activity (Fig. 3B, compare lanes 2 and 5). Thus, spacer motif) reduces processing to levels close to zero, even as in the sea urchin injection experiments described above, at relatively low molar excess (Fig. 4 A and C). This more extensive base complementarity between the U7 snRNA and effective competition of the complete wild-type competitor the histone hairpin cannot be said to be an absolutely RNA as compared to that of either conserved motif alone is essential element for 3' processing. This is supported by the consistent with the concept that both elements cooperate in finding that some processing (5-10%6) is possible in vitro even directing histone 3' end formation (6, 7). when the histone hairpin structure has been deleted (compare As seen in Fig. 3B, the mutant falsepal pre-mRNA is a poor lane 7 to the control lane 9; ref. 30). processing substrate (17-22% of wild type). From the above In the openpal construct (Fig. 3A), 10 out of 12 nucleotides results it appears possible that the processing rate for this capable of base pairing with the U7 snRNA have been mutant is reduced to -20%6 because this pre-mRNA is not preserved, but two base alterations in the stem result in a capable of binding the HBF due to the mutation of its target sequence that cannot be folded into a stem-loop structure. sequence. Ifthis is the case, processing ofthe labeled falsepal Despite the presentation of most of the potential contact sites pre-mRNA substrate should be insensitive to competition in a readily accessible linear form, processing of this precur- with a vast excess of wtpal compRNA that is presumed to sor RNA is reduced by a factor of 5 (Fig. 3B, lane 6). Hence bind the HBF. As shown in Fig. 4C, this is indeed what is it appears that both in vivo (35) and in vitro (this paper) observed. Thus these experiments suggest that the residual presentation of the wild-type sequences in a stem-loop 20% of the processing activity can be attained either by configuration is required for maximal efficiency of 3' proc- preempting the HBF with excess of wtpal compRNA or by essing, whereas deletion, mutation, or linearization of the alterations in the stem-loop sequence that prevent binding of hairpin reduces processing to 5-20% of the control value. the HBF to its target site. We designed the fstemlwtloop mutant (Fig. 3A) to inves- Interestingly, when the labeled wild-type processing sub- tigate the relative contribution of the stem sequences. While strate is challenged by an excess of fstem/wtloop compRNA, maintaining the wild-type loop nucleotides, this construct no inhibition can be observed (Fig. 4C). Apparently, more contains a false stem sequence with 12 nucleotides altered. than just the loop sequence is required for efficient recogni- This mutant supports 3' processing at a level of about 40%o tion by the HBF; rather the stem nucleotides of the wild-type (Fig. 3B, lane 4) of the control value suggesting that the hairpin appear to play a role during 3' processing, perhaps by wild-type primary sequence of the stem is important for providing necessary contact points for the presumptive HBF. maximal rate of 3' processing (see below). However, it Rescue Experiments. If the competition experiments reflect should be noted that the requirement for wild-type stem the sequestration or preemption of a factor that can then no Downloaded by guest on September 27, 2021 4348 Biochemistry: Vasserot et al. Proc. Natl. Acad Sci. USA 86 (1989)

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] PROC.E qW _ _ _NW -p 4b 1 2 3 4 1 2 3 4 5 6 complete wt comp RNA (---) wtpal comp RNA (-o-) 5 6 7 8 91011 FIG. 5. Rescue experiment. (A) HBF is Sm-precipitable. Lanes: C 1, pre-mRNA input; 2, processing in 7.5,4l ofnuclear extract; 3, same as lane 2 but in presence of 10-fold molar excess of wtpal compRNA processing (referred to as processing in preempted extract); 4, 7.5 JAIofuntreated activity extract restores processing of 7.5 of preempted extract; 5, R A t.l Hpa 100% II-digested pBR322; 6-8, each fraction tested for complementation 1 is by itself inactive in processing; 9-11, ability of these fractions to restore processing of preempted extracts. (B) HBF can be separated 80% from U7 snRNA. Lanes: 1, input RNA; 2, processing in nuclear extract; 3, processing in preempted extract; 4, Hpa IT-digested 60% :X X~~~~~~~~ pBR322; 5, restoration of processing activity by Mono Q fraction 15 40% (compare with lane 3); 6, complementation between an Sm-depleted U nuclear extract and Mono Q fraction 15. 20% activity using a column chromatography fractionation pro- 0% cedure developed by Kramer et al. (40). When Mono Q 1:0 1:1 1:3 1:10 1:30 1:100 1:300 1:1000 1:3000 fractions are assayed for their ability to restore processing in preempted extracts, the HBF activity shows a broad distri- FIG. 4. Effect of competitor RNAs on processing activity. The bution noticeably different from, but overlapping with, the competitor ratios are given in molar excess over the labeled substrate other two known processing components, the heat-labile RNA. NX, nuclear extract; Hpa marker, Hpa II-digested pBR322; factor and the U7 snRNP. A particular fraction can be Proc., processed RNA. (A) Autoradiogram of the labeled wild-type obtained in a substrate in self-competition. (B) Autoradiogram of the labeled that, complementation test, shows HBF activity (Fig. 5B, compare lanes 3 and 5) but lacks U7 snRNP (lane wild-type substrate in presence of wtpal compRNA. (C) m, Labeled wild-type substrate in self-competition; o, in the presence of wtpal 6). Furthermore, SP6 mapping failed to reveal the presence compRNA; *, in the presence of downstream spacer element comp- ofany U7 snRNA in this Mono Q fraction, demonstrating that RNA; ci, in the presence offalsepal compRNA; A, in the presence of the HBF activity does not require the presence of the U7 fstem/wtloop compRNA; A, labeled falsepal mutant substrate in snRNA to restore processing. Hence, the HBF appears to be presence of wtpal compRNA. separable from the U7 snRNP. It could represent some still unidentified snRNP or could be, at its simplest, a processing longer bind to the reporter substrate, one should be able to protein (see Discussion). restore processing by adding back HBF-containing fractions to the preempted processing extract and use this rescue experiment to characterize the relevant component(s) in a DISCUSSION preliminary way. When additional untreated nuclear extract The role of the two conserved RNA elements in producing is added to the preempted extracts, processing is restored to the mature 3' end of histone mRNAs has been extensively near normal levels, as expected (Fig. 5A, lane 4). Addition of investigated and their paramount importance unanimously the cytoplasmic supernatant (S100 fraction) has no effect demonstrated (for review, see refs. 6 and 7). The understand- (lane 11), suggesting that the HBF is a nuclear factor. Since ing oftheir respective contribution to the processing reaction the histone hairpin is known to mediate regulation of the began with the demonstration that the downstream spacer replication variant mRNA pools (37, 38) and since the heat- motif is a target for RNA-RNA duplex formation with the 5' labile factor (29) may play a role in this regulatory circuit (39), terminal sequence of the U7 snRNA (24), as proposed (22). we wanted to know whether the HBF and the heat-labile The present report focuses on the molecular mechanisms factor were one and the same. This appears not to be the case mediated by the evolutionary highly conserved hairpin struc- because nuclear extracts preheated to 50'C for 15 min [a ture (34) that is included in both histone pre-mRNA and the procedure that completely inactivates the heat-labile factor mature mRNA. but leaves the snRNPs apparently unaffected (29)] are still Our competition and rescue experiments suggest that the capable ofenhancing processing of a preempted extract (lane hairpin loop provides another anchor point for the processing 10). By contrast, nuclear extracts depleted with anti-Sm machinery. Indeed, a HBF appears to specifically recognize antibodies do not rescue processing activity (lane 9). This the histone stem-loop structure. The interaction between the leads us to the conclusion that the HBF removed during Sm inferred HBF and its RNA target is ofmajor importance since depletion carries an Sm determinant or is associated with a it contributes to as much as 80% of the processing efficiency particle (perhaps U7 snRNP) precipitable with this antiserum under the in vitro experimental conditions used. Apparently, (see Discussion). the binding of the HBF to the histone pre-mRNA can be U7 snRNA Does Not Participate in the Restoration of Pro- interfered with in one of several ways: addition of a wild-type cessing Activity in HBF-Depleted Extracts. To definitely rule hairpin competitor RNA, changes in the primary sequence, out the participation of the U7 snRNA in the observed or linearization or deletion of the HBF target site. We phenomenon, we attempted to separate it from the HBF hypothesize that in all these cases the processing apparatus Downloaded by guest on September 27, 2021 Biochemistry: Vasserot et aL Proc. Natl. Acad. Sci. USA 86 (1989) 4349 is stalled by blockage of the ability of HBF to make contacts 4. Nomura, M., Gourse, R. & Baughman, G. (1984) Annu. Rev. with the histone hairpin and this results in a loss of 80-95% Biochem. 53, 75-117. 5. Noller, H. F. (1984) Annu. Rev. Biochem. 53, 119-162. of processing rate, the residual processing activity being 6. Birnstiel, M. L., Busslinger, M. & Strub, K. (1985) Cell41, 349-359. presumably directed, in the main, by the downstream spacer 7. Bimstiel, M. L. & Schaufele, F. J. (1988) in Structure andFunction element. ofMajor andMinor Small NuclearRibonucleoprotein Particles, ed. The question remains unanswered whether the HBF is a Birnstiel, M. L. (Springer, Heidelberg), pp. 155-182. 8. Schumperli, D. (1986) Cell 45, 471-472. free entity or whether under physiological conditions it is part 9. Marzluff, W. F. & Pandey, N. B. (1988) Trends Biochem. 13, 49- of the U7 snRNP. The slightly different chromatographic 52. behavior of the HBF and U7 snRNP on a Mono Q column 10. Aziz, N. & Munro, H. N. (1987) Proc. Natl. Acad. Sci. USA 84, leads to a partial separation of these two activities (although 8478-8482. 11. Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., this could be a consequence of dissociation due to salt Rouault, T. A., Klausner, R. D., & Harford, J. B. (1988) Science concentration); thus HBF as a distinct entity, free from U7 240, 924-928. snRNP, can be recovered. Since an Sm-depleted nuclear 12. Kozak, M. (1986) Proc. Natl. Acad. Sci. USA 83, 2850-2854. extract cannot complement preempted extracts, we suggest 13. Cech, T. R. & Bass, B. R. (1986) Annu. Rev. Biochem. 55, 599-629. determinant. Mowry and Steitz 14. Hall, K. B., Green, M. R. & Redfield, A. G. (1988) Proc. Natl. that the HBF carries an Sm Acad. Sci. USA 85, 704-708. (41) have described a non-snRNP factor binding upstream of 15. Solnick, D. & Lee, S. I. (1987) Mol. Cell. Biol. 7, 3194-3198. the mouse H3 precursor processing site. This factor, present 16. Mattaj, I. W. (1988) in Structure and Function ofMajor and Minor in a fraction that contains proteins between 100 and 500 kDa Small Nuclear Ribonucleoprotein Particles, ed. Birnstiel, M. L. but does not contain (most of the) snRNPs and is anti-Sm (Springer, Heidelberg), pp. 100-114. 17. Gilmartin, G. M., Schaufele, F. J., Schaffner, G. & Birnstiel, M. L. reactive, and its binding is insensitive to prior micrococcal (1988) Mol. Cell. Biol. 8, 1076-1084. nuclease treatment (41). We do not know at present whether 18. Christie, G. E., Farnham, P. J. & Platt, T. (1981) Proc. Natl. Acad. the HBF described here and their non-snRNP factor are Sci. USA 78, 4180-4184. identical. 19. Romaniuk, P. J., Lowary, P., Wu, H. N., Stormo, G. & Uhlenbeck, 0. C. (1987) Biochemistry 26, 1563-1568. The HBF could also be a U7 snRNP protein or a factor 20. Thomas, M. S. & Nomura, M. (1987) Nucleic Acids Res. 15, 3085- associated with this particle. In this context, it is noteworthy 3096. that in sea urchin and mouse, both histone and U7 snRNA 21. Feng, S. & Holland, E. C. (1988) Nature (London) 334, 165-167. hairpins are brought in exactjuxtaposition when RNA duplex 22. Strub, K., Galli, G., Busslinger, M. & Birnstiel, M. L. (1984) EMBO J. 3, 2801-2807. formation between the downstream spacer motif and the U7 23. Vitelli, L., Kemler, I., Lauber, B., Birnstiel, M. L. & Busslinger, snRNA has occurred, even though the length of the small M. (1988) Dev. Biol. 127, 54-63. RNAs are quite divergent (22, 30, 42). This strict topological 24. Schaufele, F., Gilmartin, G. M., Bannwarth, W. & Birnstiel, M. L. relationship also brings the Sm-binding site of the U7 snRNP (1986) Nature (London) 323, 777-781. 25. Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. (1983) Nucleic in close proximity to the hairpin of the histone pre-mRNA Acids Res. 11, 1475-1489. (for illustration, see refs. 7 and 42). There is no evidence at 26. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, the moment that one of the Sm proteins of U7 snRNP is the K. & Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7056. same as the HBF we have detected. However, there are 27. Gick, O., Kramer, A., Keller, W. & Birnstiel, M. L. (1986) EMBO for snRNP proteins mediating substrate binding. J. 5, 1319-1326. precedents 28. De Robertis, E. M., Lienhard, S. & Parisot, R. E. (1982) Nature For instance, a micrococcal nuclease-treated (inactive) (London) 295, 572-577. RNase P retains its capacity to bind tRNA precursors, 29. Gick, O., Kramer, A., Vasserot, A. & Birnstiel, M. L. (1987) Proc. suggesting an important role of a protein component (43). Natl. Acad. Sci. USA 84, 8937-8940. Moreover, the 5' splice site selection seems to also require 30. Cotten, M., Gick, O., Vasserot, A., Schaffner, G. & Birnstiel, M. L. (1987) EMBO J. 7, 801-808. U1 snRNP proteins (44, 45), the intron binding protein 31. Birchmeier, C., Grosschedl, R. & Birnstiel, M. L. (1982) Cell 28, appears to be U5-associated (46, 47), and the 64-kDa protein 739-745. that recognizes the AAUAAA sequence (48) could be sn- 32. Tinoco, I., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, RNP-associated as well (49). Clearly, further characteriza- 0. C., Crothers, D. M. & Gralla, J. (1973) Nature (London) 84,498- 502. tion of the HBF is needed and will provide insights into the 33. Mohun, T., Maxson, R., Gormezano, G. & Kedes, L. (1985) Dev. exact mechanism of the processing reaction. Biol. 108, 491-502. 34. Busslinger, M., Portmann, R. & Birnstiel, M. L. (1979) Nucleic We are very grateful to Gotthold Schaffner and Klaus Kalusa for Acids Res. 6, 2997-3008. oligonucleotide syntheses, to Eddy De Robertis for his generous gift 35. Birchmeier, C., Folk, W. & Birnstiel, M. L. (1983) Cell35,433-440. of anti-Sm sera, to Octavian Gick-Schatz for providing the deltapal 36. Lynn, S. P., Kasper, L. M. & Gardner, J. F. (1988) J. Biol. Chem. the of a T7 263, 472-479. and Cpal constructs, to Martin Nicklin for gift powerful 37. Luscher, B., Stauber, C., Schindler, R. & Schumperli, D. (1985) polymerase, and to the members of the Service Department of the Proc. Natl. Acad. Sci. USA 82, 4389-4393. Research Institute of Molecular Pathology for sequencing. We also 38. Stauber, C., Luscher, B., Eckner, R., Lotscher, E. & Schumperli, thank Matt Cotten for his valuable help and advice and Margaret D. (1986) EMBO J. 5, 3297-3303. Chipchase and Meinrad Busslinger for critical reading of the manu- 39. Luscher, B. & Schumperli, D. (1987) EMBO J. 6, 1721-1726. script. The EB1 mouse cells were used with the kind permission of 40. Kramer, A., Frick, M. & Keller, W. (1987) J. Biol. Chem. 262, Peter Swetly (Bender, Vienna). We thank Ingeburg Hausmann and 17630-17640. Fritz Ochsenbein for the preparation of the figures, and Marianne 41. Mowry, K. L. & Steitz, J. A. (1987) Science 238, 1682-1687. Vertes for secretarial help. This work was supported by the Research 42. Soldati, D. & Schumperli, D. (1988) Mol. Cell. Biol. 8, 1518-1524. 43. Nichols, M., So6l, D. & Willis, I. (1988) Proc. Natl. Acad. Sci. USA Institute of Molecular Pathology and in part by grants of the Swiss 85, 1379-1383. National Science Foundation and the Kanton of Zurich. This work 44. Mount, S. M., Pettersson, I., Hinterberger, M., Karmas, A. & was partial fulfillment of the Ph.D. requirements for A.P.V. and for Steitz, J. A. (1983) Cell 33, 509-518. F.J.S. who was supported by a postgraduate scholarship from the 45. Zhuang, Y. & Weiner, A. M. 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