Finding functions for small nuclear RNAs in yeast

430

TIBS 11 - October 1986

Finding f u n c t i o n s R

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Small nudear RNAs (snRNAs) have been suggested to mediate a variety of steps in gene erpresdon, with an empha~ on essential R N A processing reactions. By using the yeast Saccharomyces cerevisiae, in w h ~ genetic and biochemical approaches can be coupled, it is now apparent #rot the ways in which snRNAs contribute to the growth of even a simple eukaryote are also likely to be more diverse, and possibly more subtle, than previously

/magined. The 'U1 hypothesis': a popular prototype It is now almost two decades since the discovery of small nuclear RNAs (snRNAs) in mammalian cells (see Ref. 1). Early biochemical studies revealed that these ubiquitous and highly abundant molecules are uridine-rich (thus their designation as U 1 . . . U6), metabolically stable, terminated by a unique 5' cap structure 2,2,7-trimethylguanosine (pm2,2,7G), and housed in ribonucleoprotein particles (snRNPs). However, it was not until the discovery some ten years later 2 that sera from patients with the autoimmune disease systemic lupus erythematosis (SLE) could precipitate these RNP particles from species as distantly related as insects, that their remarkable evolutionary conservation was appreciated. This observation provided a compelling basis for the general argument that snRNAs play functionally conserved, and thus presumably important, roles in eukaryotes. In particular, it was pointed out 3 that the first 18 nucleotides of U1 were complementary to the consensus sequences at the 5' and 3' splice junctions of premRNAs, offering a simple and elegant solutiontotheenigmaofhowintronborders are identified and precisely juxtaposed. Although this specific model has required modification (see below), the 'U1 hypothesis' none-the-less provided an irresistible paradigm, and a broad spectrum of RNA processing reactions is now thought to be mediated by complementarities between sequences within particular snRNAs and target sequences in the precursor RNA substrates, Despite the enormous popular appeal of these models, experimental proof has been hard to come by. Useful in vitro systems for mRNA splicing and polyadenylation have become available only

recently and are not likely to be readily forthcoming for rRNA processing, for example. Moreover, tractable genetic systems are lacking among the higher eukaryotes where the majority of interest has been focused. Appreciating the tremendous advantage of being able to combine classical and modem genetics with traditional biochemical approaches, we turned several years ago to the yeast Saccharyomyces cerevisiae, At that time there was some question as to whether lower eukaryotes even had snRNAs; none had been described, and SLE sera such as 'anti-Sm' failed to precipitate labelled RNAs from yeast or Dictyostelium 2. The initial challenge was thus to identify the yeast analogues and clone thegenes,

The snRNA familyin yeast: unexpectedly large and diverse Following unsuccessful efforts to detect meaningful signals by northern or Southern hybridization using heterologous snRNA clones, we resorted to direct analysis of in vivo 32p-labelled RNAs, guided by a set of criteria drawn from metazoan snRNAs. In particular, we focused on stable RNAs in the size range of =100-300 nucleotides. The most powerful diagnostic marker was the presence of the trimethylguanosine 5' cap, a unique hallmark of snRNAs, which we identified by its characteristic enzymatic digestion patterns. As we have described4, this exercise revealed a number of candidate species, each with a surprisingly low abundance. We estimated that an 'average' yeast snRNA was present in the order of 20(0500 molecules per haploid cell, in striking contrast to the million molecules of U1, for example, in a HeLa cell. Further attempts to establish the complete set size of the yeast snRNA family, as well as its more detailed biochemical characterization, were severely hindered c. Guthrieis at the Deparanentof Biochemistryand by the difficulty of obtaining pure prepBiophysics, Universityof California, San Francisco, arations in sufficient yields. A major San Francisco,CA94143, USA. breakthrough occurred when we were ~ 1986, Elsevier Science Publishers B.V., Amsterdam

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snRNAsabl to imemunopreci antipserum itateusinthgedirectedYeast against the trimethylguanosine cap 5 (a generous gift from the laboratory of J . A . Steitz). Gel fractionation of immunoprecipitated snRNAs 6, in conjunction with cloning and hydridization analyses (Refs 3, 4 and 6; Parker et al., unpublished), allows us to draw the foilowing conclusions. (1) There are about 24 unique snRNAs in S. cerevisiae; this is likely to be a minimum estimate. (2) While the 'average' snRNA numbers several hundred molecules per cell, there is a subset of'minor' species which are present in the order of tens of molecules per cell. (3) At least four yeast snRNAs (snR17-20, by our nomenclature6) appear to be larger than 300 nucleotides, and one of these (snR20) exceeds 1000 nucleotides. Each of these results stands in dramatic contrast to the current picture of metazoan snRNAs, which is dominated by a small group (U1-U6) of very abundant snRNAs (200 000-1 000 000 molecules per cell), the largest of which (U3) is only 216 nucleotides long. What is the biological significance of these apparent disparities? Is it reasonable to think that a comparatively simple, unicellular eukaryote has a more complicated array of snRNAs than the developmentally complex metazoans? What, in any event, are all of these RNA species doing? Their number considerably exceeds the number of (unique) flmctions currently proposed for them. (At least)six snRNAs are non-essential The central premise of our approach has been that snRNAs perform essential RNA processing reactions and are thus of required for growth. The generation of conditional mutations would then allow the identification of the specific biochemical defect resulting from the absence of that snRNA function under non-permissive conditions. The success of this strategy requires that the snRNA of interest be encoded by one (or no more than a few) gene(s), and that the gene(s) be essential for growth. As a first step, it was encouraging to find that the genes encoding the eight yeast snRNAs we have tested to date are encoded by single copy genes (Refs 4, 7 and 8; Parker et al., unpublished). To assess the essentiality of a given snRNA, a gene replacement operation is performed using the scheme pioneered by Rothstein and colleagues9 (Fig. 1). First, the gene is disrupted in vitro

T I B S 11 - O c t o b e r 1986

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Fig. 2. A simplified view of the mRNA splicingpathway. Conserved sequences within the introns o.fyeast pre-mRNAs are indicated," the functional equivalents of these regions are alsofound in otherfungi and in metazoans but the sequences are less well conserved (see Fig. 3). These elements are thought to be binding sitesfor snRNPslz-14, which mediate the higher-orderfolding of the intron necessaryfor catalysis (seeR ef 11). When radioacavely.labeUedprecursor is used, these in vitro-assembled complexes, and the so-called lariat intermediates formed by cleavage at the 5' junction, can be identified as rapidly sedimenting forms in sucrosegradients (Ref. 15). The disassembly of this complex upon the completion of the splicing reaction is not at all understood. For technical reasons we are unable to reproduce this figure in colour. See the October issue of Trends in Biochemical Sciences for full colour illustration.

432 (Fig. la) by replacing a region of the coding sequence with a DNA fragment containing a selectable marker. The interrupted, non-functional gene is then reintroduced into the chromosome (Fig. lb), precisely replacing the resident chromosomal copy. Because haploid cells lacking a functional SNR gene are expected to be inviable, the replacements are performed in a diploid cell. The heterozygous (SNR/snr) transformants are then sporulated (Fig. lc). If an essential gene has been successfullyinactivated, each tetrad should give rise to only two viable spores, both of which should contain the non-disrupted gene (and can be easily recognized as such because they lack the inserted selectable marker). This initial foray into 'gene killing'7 m e t - once again- with completely unexpected results: the deletion of the single copy gene for SNR3 has no detectable effect on the growth of haploid cells under a wide variety of conditions tested, Even when sister spores were co-cuP turedinachemostatundernon-selective conditions for more than 60 generations, spores containing a functional copy of the SNR3 gene exhibited no competitive advantage! In an attempt to reconcile this result with our anticipation that all snRNAs would be essential, we suggested 7 that some yeast RNAs might perform redundant or partially overlapping functions. A hit-or-miss approach to identifying functional substitutes would prove to be a tedious proposition, given what we now know to be the large number of yeast snRNAs. Initial efforts were focused, therefore, on snRNAs which exhibit similar physicalproperties; for example, snR4 and snR5 co-migrate with snR3 in two-dimensional polyacrylamide gel electrophoresis under seminative conditions6. We have since shown that, individually, each of these genes is completely dispensable, as are the genes for snR8 and snR9 (Ref. 6; Parker et al., unpublished). In fact, even the quintuple mutant snr3: :LEU2, snr4: :URA3, snr5: :TRP1, snr8: :HIS3, and snr9: :URA3isviable! Thus, it was a relief to find8 that disruption of the gene encoding snR10 at least impairs growth, particularly at elevated osmotic strengths or low temperatures. At 25°C the doubling time ofsnrlO strains is 50% greater than that of otherwise isogenic sister spores. The conditional nature of the phenotype is surprising as the loss of the SNRIO gene product is, of course, complete and non-conditional. It is interesting to note that, as first discovered many years ago 10, coldsensitive mutants in bacteria are fie-

TIBS 11 - October 1986

quently defective in ribosome assembly, Indeed, Tollervey (unpublished) has observed an alteration in the rRNA processing pattern of snrlO ceils: the 35S rRNA precursor is cleaved at a reduced rate, and by a pathway used only rarely in wild-type strains. Additional data from ToUervey suggest the possibility that other 'dispensable' snRNAs play a role in rRNA processing in that they behave as if hydrogen-bonded to particular rRNA precursors. We are now testing this hypothesis by analysing the processing profile in the sextuple mutaut; the strain is viable (Parker et al., unpublished), although it may be slightly more growth-impaired than the single mutant lacking snR10, The dispensability of yeast snRNA genes may indeed be the rule. Three other examples have recently been seen by Fournier (Amherst) and by Cesareni (EMBL) (pers. commun.), although the specific relationship between the RNAs studied in each of our laboratories has yet to be established. Conceivably, the apparent dispensability of perhaps a quarter of the snRNAs in S. cerevisiae will be attributed to their partially redundant or overlapping roles in a highly complex pathway, such as that of ribosome assembly. An alternative possibility is also compatible with the existing data: the participation of snRNAs in a given process may not be an essential contribution, but rather influence the choice and/or efficiency of utilization of a particular pathway under a particular set of conditions. A test of this provocative hypothesis must first await identification of all the snRNAsinvolved in that process, Other models can also be entertained, It is worth pointing out that this is not an isolated example of an apparently conserved biological phenomenon whose function is not obvious at the phenotypic level. We have commented previously7 on the widespread dispensability of modifications in tRNAs; acetylation of histones may comprise another such paradox. More recent examples are provided by the kind of approach we are taking in yeast; the dispensability of heatshock proteins is a good case in point, Thus the assignment of function to evolutionarily conserved structures or processes, contrary to expectation, appears to be a general problem in biology. snRNAs implicated in splicing are essential In the meantime, the challenge is to identify the functional counterparts of RNAs believed to play essential roles in

mRNA splicing. A composite view of this pathway is depicted in Fig. 2. Three regions of the intron are required for accurate and efficient splicingn. In S. cerevisiae these elements show virtually complete conservation of primary sequence, while they exhibit increasing degeneracy in other fungi and in metazoans, respectively (Fig. 3). From recent experiments in mammalian in vitro systems, it now appears that these regions serve as binding sites for specific snRNPs, U1 at the 5' junction12, U5 (probably) at the 3' junction13, and U214 upstream of this region, at the position termed the 'TACTAAC box' in yeast. It is a reasonable conjecture that the splice sites are positioned via the interactions of the snRNPs with the substrate and with one another. This is consistent with the observation of a rapidly sedimenting complex termed the spliceosome 15, which contains the intermediates of the two-stage splicing reaction- the 5' exon, and the so-called lariat structure generated when the 5' end of the intron forms an intramolecular branch via a 2'-5' phosphodiester bond with the adenosine residue at the 3' end of the T A C T A A C box 16. This complex (40S in yeast) presumably contains the snRNPs, as well as one or more hnRNPs 17. One approach to seeking out the splicing snRNAs is thus to exploit the existence of the yeast in vitro system TM, and ask which snRNA species are enrichedin fractions shown to be essential for splicing. A complementary strategy is based on identifying homologies to the URNAs. To date, all metazoan snRNAs implicated in the maturation of premRNA have been shown to possess a common structural motif, the sequence A(Ua_6)G embedded in a singlestranded region, which is the binding site for a complex of four proteins that cornprise the 'Sm' antigenic determinant ~9. None of the six dispensable yeast snRNAs contain this motif (Refs 7 and 8; Parker et al., unpublished). In striking contrast, the first essential snRNA we identified, snR7 (Ref. 5; Patterson and Guthrie, unpublished), contains the sequence A U t G in what is very likely to be a single-stranded domain. We thus hypothesized that a subset of yeast snRNAs are of the 'Sm-type', but that the antigenic determinant has diverged too far to be recognized by the human sera. To test the model that the sequence in snR7 is a functional binding site: can snR7 become Sm-precipitable after injection into Xenopus eggs or oocytes, which are known to stockpile free snRNP proteins early in development19?

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T I B S 11 - October 1986

This was, indeed, found to be so (Riedel et al., submitted), and at least one other species, designated snR14, also became Sm-precipitable. Sequence analysis of snR14 has since revealed an excellent candidate for the Sm binding site: AUsG. A gene disruption of S N R 1 4 has now shown that this single copy gene is also essential (Siliciano and Guthrie, unpublished), These experiments are exciting for a number of reasons. Among these is the opportunity to gain the first direct handle on the snRNP proteins in yeast. While their apparent antigenic divergence prevents use of SLE sera for the isolation of the Sm-analogues in S. cerevisiae, proteins which are predicted to bind to the respective sites in snR7 and snR14 can now be looked for. In addition to this biochemical approach, point mutations can be made in these presumptive binding sites; second-site revertants to such mutations are likely to genetically identify the yeast Sm-counterparts. The virtue of this strategy derives from the use of such mutants for the identification, by sequential suppressor isolations, of the other snRNP proteins which, by analogy to work in metazoans, must interact closely with these core proteins, Given that snR7 and snR14 are essential and are apparently of the Sm-type, can evidence that they are indeed involved in splicing now be provided? The lethal phenotype of the null alleles requires the generation of conditional alleles in order to determine the biochemical basis of lethality in vivo. As a first step, we have succeeded in engineering the conditional synthesis of snR7. As illustrated in Fig. 4, this can be done by fusing the upstream control elem e n t of t h e yeast G A L l gene to the cod-

ing sequences of S N R 7 , allowing trans- at least a portion of this homology are cription of snR7 to be regulated by the the same size as vertebrate U2, i.e. =200 choice of carbon source: 'on' with galac- nucleotides (Riedel et al., unpublished; tose and 'off' with glucose. Cells in which Tollervey and Mattaj, unpublished). It is this contruction, carded on an autonom- interesting to speculate that the stricter ously replicating plasmid, provides the sequence requirements of the S. cereonly source of snR7 die approximately visiae splicing machinery (cf. Fig. 3) six generations after a shift from galac- reflect a greater reliance on RNAtose to glucose. This lag is presumably mediated aspects of the mechanism. due to the time required for dilution of the stable snR7 molecules synthesized Conclusions and speculations during growth on galactose. The effiThe demonstration that yeast has an ciency of splicing of various pre-mRNAs unexpectedly large number of snRNA can be assayed by performing northern types, many of which are apparently dishybridization and primer extension pensable, has important ramifications analyses on RNA extracted from cells for the conceptual understanding of depleted for snR7. In all cases tested, snRNA function. In considering these, there is a marked accumulation of the frst question which inevitably arises unspliced precursor RNA (Patterson is whether or not this situation is likely to and Guthrie, unpublished). Similar be peculiar to yeast. While the existence studies are underway with snR14, of certain 'fungal-specific' snRNAs is Finally, experiments performed in col- certainly a reasonable (and interesting) laboration with Abelson's group (Riedel possibility, several findings favour the et al., unpublished) show that each of prediction that the snRNA profile of these RNAs is highly enriched in Frac- higher eukaryotes will also turn out to be tion I of the in vitro splicing system20. more complex than has been assumed. The activity of this fraction is sensitive to First is the recent discovery (in sea urchin treatment with micrococcal nuclease or and mammals) of several hitherto with antibody to the trimethylguanosine unknown snRNAs, each present at 1/10 cap 2°. The RNAs also appear to co-frac- to 1/30 the abundance of U1-U62122. In tionate with the spliceosome complex, as addition, Tollervey and Mattaj (unpubdoes the much larger RNA, snR20. So lished) have shown that the complexity now the final mystery: which are the of snRNAs we observe in S. cerevisiae is functional analogues of U1 etc., and pre- mirrored not only in other fungi, but in cisely what roles are snR7, snR14, and the pea plant as well. As we have snR20 playing? Ares (unpublished) has observed with S. cerevisiae, only a small recently made the astonishing observa- subset of the 20 or so snRNAs found in tion that S. cerevisiae contains a these other organisms become Sm-presequence with 285% homology to ver- cipitable after injection into X e n o p u s tebrate U2, embedded in an RNA of oocytes. In markedcontrasttothesitua---1200 nucleotides. This single copy tion in S. cerevisiae, however, the Smgene, which appears to encode snR20 class species in other fungi are signific(our nomenclature6), is essential for via- antly more abundant than the non-Sm bility. In other fungi, RNAs which share type for the same organism. Interest-

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Fig. 4. A method for assaying the phenotype of an essentialgene. Thegeneofinterest(inthiscase, SNR7, which encodes an snRNA essential for growth) is cloned behindaninduciblepromoter (in thiscase, the upstreamcontrolregionfortheyeastGALlgene)and introduced into a cell in which the chromosomalcopy of the SNRgene has been disrupted as in Fig. 1. Cells will grow normally in galactose, since the promoter is active. When cells are shifted to glucose, transcription from this promoter is turned off. Cells grow at the wild-type rate for. six generations after the shift, presumably the time it takes to dilute out the snR7 molecules synthesized during growth in galactose. The biochemical phenotype of cells depleted for snR7 can thus be assayed by preparing RNA from cells shifted for varying Omes and performing northern hybridization or primer extension analyses on transcripts of interest.

ingly, it also appears that the extent of m R N A splicing in other fungi is much greater than in S. cerevisiae; that is, a greater n u m b e r of genes contain introns, and spliced genes frequently contain m o r e than one intron. T h e most likely unifying m o d e l at this time, while admittedly p r e m a t u r e , would be that all eukaryotes will be found to contain a diverse set o f s n R N A s . Precise n u m b e r s may well vary significantly according to organism, cell type, etc., but an 'average' value might be several dozen. Perhaps the m o r e striking difference will turn out to be the relative abundance of the different s n R N A s to one another, which will presumably reflect both the scope o f their R N A 'targets' (e.g. some, like U721), may perform gene-specific functions 6, as ~vell as the nature of their participation in a given process (e.g. many s n R N P com?onents of the spliceosome might be viewed as stoichiometric). Until now the most 'visible' s n R N A s have b e e n the Sm-class, whose function may be restricted to m R N A processing. Sac~ h a r o m y c e s cerevisiae, in which splicing does not appear to be a p r e d o m i n a n t activity, may thus provide the most 'balanced' view of s n R N A function in toto. T h e apparent dispensability of perhaps the majority of the s n R N A s in this organism forces us to reconsider our general prejudices about the ways in which s n R N A s contribute to the growth of eukaryotes; conceivably these are m o r e highly specialized, or possibly m o r e subtle, than previously imagined.

References 1 Busch, H., Reddy, R., Rothblum, L. and Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-653

2 Lemer, M. R. and Steitz, J. A. (1979) Proc. Natl Acad. Sci. USA 76, 5495-5499 3 Lerner, M. R., Boyle, J. A., Mount, S.M., Wolin, S. L. and Steitz, J. A. (1980) Nature 283,220-224

4 Wise, J.A., Tollervey, D., Maloney, D., Swerdlow, H., Dunn, E. and Guthrie, C. (1983), Cell35, 742-751 5 Guthrie, C., Riedel, N., Parker, R., Swerdlow, H. and Patterson, B. (1986) Yeast Cell Biology (UCLA Symposia on Molecular and Cellular Biology,New Series, Vol. 33), pp. 301-321, Alan R. Liss 6 Riedel, N., Wise, J. A., Swerdlow, H., Mak, A. and Guthrie, C. Proc. Natl Acud. Sci. USA

(in press) 7 Tollervey, D., Wise, J. A. and Guthrie, C. (1983) Cell35, 753-762 8 Tollervey, D. andGuthrie,C.(1985)EMBOJ. 4, 3873-3878 9 Rothstein,R.J.(1983)MethodsEnzymol. 101, 202-211 10 Guthrie, C., Nashimoto, H. and Nomura, M. (1969) ColdSpring HarborSymp Quant. Biol. 34, 69-75 11 Vijayraghavan, U., Parker, R., Tamm, J., Imura, Y., Rossi, J., nbelson, J. and Guthrie, c. (1986) EMBO J. 5, 1683-1695 12 Mount, S. M., Patterson, I., Hinterberger, M., Karmar, A. and Steitz, J. A. (1983) Cell 33, 509-518 13 Chabot, B., Black, D. L., LeMaster, D. M. andSteitz, J. A. (1985) Science 230,1344-1349 14 Krainer, A. R. and Maniatis, T. (1985) Cell42, 725-736 15 Brody, E. and Abelson, J. (1985) Science 228, 963-966 16 Domdey, H., Apostol, B., Lin, R-J., Newman, A., Brody, E. and Abelson, J. (1984) Cell 39, 603~10 17 Choi, Y., Grabowski, P., Sharp, A. and Dreyfuss, G. (1986) Science 231,1534-1539

18 Lin, R. J., Newman, A. J., Cheng, S-C. and Abelson, J. (1985)J. Biol. Chem. 260, 1478014792 19 Mattaj, I. W. and DeRobertis, E. (1985) Cell 40, 111-118 20 Cheng, S-C. and Abelson, J. (1986) Proc. Na Acad. Aci. USA 83, 2387-2391 21 Birnstiel, M. L., Busslinger, M. and Strub, K. (1985) Cell 41,349-359 22 Reddy, R., Henning, D. and Busch, H. (1985) J. Biol. Chem. 260, 10930-10935

For technical reasons we are unable to reproduce this figure in colour. See the October issue of Trends in Biochemical Sciences for full colour illustration.