RNA splicing in yeast mitochondria: Taking out the twists

WONITOR 11 Sive, H.L. and Roeder R.G. (1986) Proc. Natl Acad. Sci. USA 83,

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6382~5386 Singh, H., Sen, R., Baltimore, D. and Sharp, EA. (1986) Nature 319, 154--158 Fletcher, C., Heintz, N. and Roeder, R.G. (1987) Cell 51,773-781 Sturm, R., Baumruker, T., Franza, B.R. and Herr, W. (1987) Genes Dev. 1, 1147-1160 Sturm, R. A., Das, G. and Herr, W. (1988) Genes Dev. 2, 1582-1599 MOiler, M.M., Ruppert, S., Schaffner, W. and Matthias, R (1988) Nature 336, 544-551 Clerc, R.G. et al. (1988) Genes Dev. 2, 1570-1581

18 Scheidereit, C. el al. (1988)Nature 19

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336, 551-557 Herr, W. et al. (1988) Genes Dev. 2, 1513-1516 LaBella, F., Sive, H.L., Roeder, R.G. and Heintz, N. (1988) Genes Dev. 2, 32-39 Pruijn, G0.M, van Driel, W. and van der Vliet, P.C. (1986) Nature 322,656-659 Wirth, T., Staudt, L. and Baltimore, D. (1987) Nature 329, 174-178 Dreyfus, M., Doyen, N. and Rougeon, F. (1987) EMBOJ. 6, 1685-1690 O'Hare, R, Goding, C.R. and Haigh, A. (1988) EMBO.L 7, 4231-4238

2 5 Chiu, E. etal. (1988) Cell 54,

541-552 26 Tanaka, M., Grossniklaus, U., Herr, W. and Hernandez, N. (1988) Genes Dev. 2, 1764-1778 27 Desplan, C., Theis, J. and O'Farrell, P.H. (1988) Cell 54, 1081-1090 2 8 Hoey, T. and Levine, M. (1988) Nature 332,858-861 2 9 Ko, H-S., Fast, P., McBride, W. and Staudt, L.M (1988) Cell 55, 135-144 30 Thali, M. etal. (1988) Nature 336, 598---601 31 Sigler, RB. (1988)Nature 333, 210-212 32 Gerster, T. and Roeder, R.G. (1988) Proc. Natl Acad. Sci. USA 85, 63474351

RNA SPLICINGin YEASTMITOCHONDRIA:TAKINGOUT the TWISTS L.A. GRIVELLAND R.J. SCHWEYEN* SECTIONFORMOLECULARBIOLOGY,DEPARTMENTOF MOLECULARCELtBIOLOGY,tNIVERSlTYOF AMSTERDAM,KRUISLAAN318, 10988MAMSTERDAM,THE NETHERLANDSAND*INSTITUTEFORGENETICS,UNIVERSITYOFVIENNA,AETHANSTRASSE14, A-1090VIENNA,AUSTRIA. b a c k in 1978, Slonimski and his co-workers applied the term mosaic to describe the organization of the genes coding for cytochrome b and subunit I of cytochrome c oxidase in yeast mtDNA 1. The organization was remarkable indeed, since at a time when the molecular biological world was adjusting to the idea of split genes and RNA splicing, it was found that these genes not only contained introns, but many of the introns coded in their turn for proteins. Some of these proteins, called RNA maturases, are n o w k n o w n to be required for the splicing of the intron that encodes them. Splicing is thus neatly autoregulated; the proteins control their own synthesis by destroying the mRNA that produces them (reviewed in Ref. 2). Today, some ten years later, the situation appears more rather than less complex. On the one hand, many mitochondrial introns have turned out to possess self-splicing activity. When intron-containing precursor RNAs are incubated i n vitro, the introns are excised rapidly and accurately in the total absence of protein3. On the other hand, systematic genetic screening has implicated several additional proteins in mitochondrial splicing in vivo (reviewed in Ref. 4). These proteins are e n c o d e d by nuclear genes and have to be imported into

the mitochondrion. Some of them appear to be required for only a single intron, while others have been implicated in the splicing of several introns; these introns may be removed by the same or different types of splicing mechanism. As yet, little is k n o w n about the role played by either maturases or these additional proteins. For the product of the nuclear C B P 2 gene, a protein specifically required for splicing in vivo of the self-splicing bI5 intron in the cytochrome b gene, recent work carried out in Tzagoloff's laboratory has shown that the protein can bind to the intron and modify its in vitro catalytic properties, while leaving the basic transesterification mechanism of RNA catalysis unaltered (A. Gampel, M. Nishikimi and A. Tzagoloff, submitted). Speculations on the possible functions for this and other proteins might therefore include stabilization of correct intron folding, prevention of aberrant side reactions, or enhancement of the efficiency of RNA catalysis by shielding and/or activation of reactive groups. The characterization of yet another nuclear-encoded protein required for the splicing of a number of mitochondrial introns is reported in a recent paper by SGraphin et al.5, and their findings provide grounds for speculations in "rig FFBRt:AR',"1989 VOI.. 5 NO. 2

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a new and interesting direction. These authors have identified the nuclear gene M S S l l 6 by transformation-complementation of a splicing-deficient mutant isolated some years ago by Tzagoloff and his coworkers 6. This mutant is blocked in the excision of several group I and II introns, located in the genes coding for cytochrome b and cytochrome c oxidase subunit I. DNA sequence analysis of the gene reveals that the M S S l l 6 gene product is a protein of 664 amino acids that is remarkably similar to a set of proteins that includes the eukaryotic initiation factors eIF4A1 and 4A2, the human nuclear p68 protein, and the uvrD, rep, recB and UL5 replicative proteins. The c o m m o n factor linking these proteins is that they all possess, or are thought to possess, helicase or unwindase activity. The MSSI16 product may therefore help drive splicing by catalysing folding-unfolding cycles that lead to an RNA molecule that is active in self-splicing. Alternative explanations are also possible, however, and to evaluate these it is necessary to examine some of the possible pitfalls inherent to this type of analysis. (1) Through their dependence on maturases, the splicing of many mitochondrial introns is intimately tied up with mitochondrial

WONITOR (Ref. 5 and G. Faye, pers. translation. Any interference commun.). with this process, either Whether the M S S I I 6 directly through a change in product promotes splicing a component of the transdirectly or indirectly via lational machinery, or intranslation is less clear. The directly through a reduction finding of S6raphin et al. s in overall mitochondrial that splicing of the intron energy level, will affect the bll. which is not dependent synthesis of maturases and on a maturase, is reduced by hence impede splicing. Unwindase? a mutation in MSSll6, is A further complicating Precursor interpretable in terms of a factor is that the same proRNA Mature direct effect. However, lowtein may be involved in mRNA ered energy levels, caused both processes. The leucylby reduced synthesis of tRNA synthetase, encoded components of tile respiraby the NAM2 gene in yeast:' tory chain, could have simiOther and the tyrosyl-tRNA synnuclear-encoded lar effects. In this context, it thetase encoded by the proteins is important to note that c r t l 8 gene in Neurospora MSS116 disruptants seem to crassaS are just such prodisplay reduced levels of teins. In the case of NAM2. ,~TGIil the mutations in the ~EI.F-SPLICING OF MITOCIK)NI)RIAL INTRONS IS DI'PENI)ENT ON COI'~ synthesis of proteins enaminoacyl-tRNA synthetase RECF FOI.I)ING. FOR TIIOSE INTRONS I H K I ARE ALSO DEPENDENT ON coded by at least two genes suppress splicing deficiency A blATtTRASE FOR TIIEIR EXCISION, ADDITIONAl. PROTEINS, PERIa!APS IN that lack introns. Interestingly, cytoduction caused by lack of an active CONJUNCTION WITIt TIlE MATURASE ITSELF, MAY IIELP T]tE 1NTRONof mtI)NA devoid of all CON~IAININ(; PRECURSOR RNA 1 o MAKE TIlE CONFORMKIIONA[. TRANmaturase encoded by an mitochondrial intron in the mitochondrial SITION FROM TRANSL4.'IABLE MRNAr o CKIAI.YTIC I N I I . T i l t PROD- 13 known gene for cytochrome b. ICI" OF TIlE MSSl16 GENE MAY I)RI\E Till" SPI.ICIN(; REACIION BY introns ~3 into a strain car WCA1AI.YSING FOLI)ING-UNFOI.D[NG C'ICIES TtlKI' LI:A[) riO AN RNA ing an M S S l l 6 disruption Others affect tRNA acylation MOI.ECULt{ '.X r l l t AN A(711VE CONFORMKI'ION. does not restore respiratory." and hence protein syncompetence. Although such thesisZ so that in time, the a mtI)NA could conceivably conthese cases, inactivation of the processing of many precursor tain as yet unidentified introns, this chromosomal copy of the cloned RNAs would be expected to be observation is more likely to be an halted as a consequence of im- sequences fails to reproduce the indication that the MSSI16 gene original splicing-defective mutant paired synthesis of intron-encoded has multiple functions in mitochonphenotype. maturases. driat biogenesis. (3) If indirect effects are opera(2) The sequences cloned may As yet, nothing is known about tive, the protein encoded by the not always correspond to the gene the location of the MSSll6 product defined by the original mutation. In cloned sequence need not have a in the cell. The predicted amino mitochondrial location. The T I F the complementation assays usually terminus of the protein is, howgenes recently characterized by employed to screen for suppresever, rich in serine, threonine and Linder et al. 1" are g o o d examples sion of splicing deficiency, restorbasic amino acids, features that are of this. These genes turn out to ation of even low" levels of splicing comnlon to the amino-terminal will give rise to respiratory." suf- code for the yeast cytoplasmic initicleavable presequences of many ation factor elF4A, yet when presficiency. Positive cokmies can thus imported mitochondrial proteins. A ent in high copy number, they arise not only by direct restoration mitochondrial location seems likely, of mitochondrial splicing activity, allow the suppression of missense but this has to be verified directly. mutations in the mitochondrial but indirectly, perhaps by changes Where do these findings leave gene for subunit III of cytochrome in translation or energy metus? They are significant in at least c oxidase. The mechanism of this abolism, or perhaps by compenthree ways. First, identification of sato U changes in the synthesis or effect is completely unknown. the MSSll6 protein itself opens the H o w do the results obtained by import of other proteins involved way to its purification, direct assay S4raphin et al. s measure up to in the splicing reaction. of unwindase activity and use in the above considerations? First, Such effects may account for hz vitro assays of splicing activity. the fact that in several cases, in although no comparison of wildSecond, if proven, the involvetype and mutant sequences has yet particular those in which the gene ment of an unwindase in mitobeen performed, the sequence studied was acting as a suppressor chondrial splicing would provide cloned seems likely to be that of of intronic mutations to splicing further indications of anak)gies the M S S l l 6 gene. The phenotype deficiency, the complementing between these basically rather simof a gene disruptant corresponds sequence was effective only when ple reactions and those mediated closely to that of the original present on a multicopy plasmid. by spliceosomes in the nucleus. This presumably reflects a need for mutant and a single copy of the During nuclear splicing, U4 and U6 M S S I I 6 gene is sufficient to comgross overproduction of the protein snRNAs have been shown to follow pensate for the effects of disruption inw~lved (see Refs 10, 11). In all ILK;FI{BRI]ARY1989 VOL. "5 NO. 2

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WONITOR a cycle of association and dissociation, and it has been suggested that a helicase may play a role in melting these molecules14,15. Third, should the MSSll6 protein also turn out to be involved in mitochondrial translation, it would raise some interesting questions regarding the possible mutual interd e p e n d e n c e of splicing and translation. It should be borne in mind that translation of a reading frame in an intron and splicing are temporally exclusive events, since passage of ribosomes will prevent the folding required to produce a catalytic centre. Unwindase, perhaps in conjunction with maturases, might therefore play a key role in directing the transition of those introns that are translated from their role as mRNAs to a role as catalytic units (Fig. 1). Support for such an idea also comes from a number of early observations, based mainly on the use of specific inhibitors of mitochondrial protein synthesis. These have pointed to the possible existence of links between translation and splicing16,17; pausing or stalling of ribosomes may interfere with secondary structural alterations

necessary for splicing to occur. Alternatively, the arrest of translation may sequester factors used in both processes, such as some aminoacyl-tRNA synthetases and possibly the MSS116 product. Further investigation of these observations in the light of the present findings could lead to the unravelling of some interesting principles.

aCKNOWLEDGEMENTS We are grateful to Drs G. Faye, P.P. Slonimski and A. Tzagoloff for making information available to us before publication. L.A.G. acknowledges support from the Netherlands Organization for the Advancement of Pure Research (NWO), under the auspices of the Netherlands Foundation for Chemical Research (SON). R.J.S. was supported by the Austrian Foundation for Advancement of Science (FWF).

rEFERENCES 1 Slonimski, E et al. in Biochemistry and Genetics of Yeast (Bacila, M. et al., eds), pp. 339-368, Academic Press 2 Grivell, L.A., Bonen, L. and Borst, P. (1983) in Genes: Structure and Expression (Horizons in BiochemistO, and Biophysics 17ol. 7) (Kroon,

A.M., ed.), pp.279-306, John Wiley 3 Tabak, H.E and Grivell, L.A. (1986)

Trends Genet. 2, 51-55 4 Grivell, L. A. EurJ. Biochem. (in press) 5 S6raphin, B., Simon, M., Boulet, A. and Faye, G, (1989) Nature 337, 84-87 6 Tzagoloff, A., Akai, A. and Needleman, R. (1975)J. Biol. Chem. 250, 8228-4235 7 Herbert, C.J., Labouesse, M., Dujardin, G. and Slonimski, P.P. (1988) EMBOJ. 7, 473-483 8 Akins, R.A. and Lambowitz, A.M. (1987) Cell 50, 331-345 9 Tzagoloff, A. et al. (1988) J. Biol. Chem. 263, 850-856 10 Schmidt, C., SOllner, T. and Schweyen, R.J. (1987)Mol. Gen. Genet. 210, 145-152 11 Ben Asher, E. et al. Mol. Gen. Genet. (in press) 12 Linder, P. and Slonimski, P.E Proc. Natl Acad. Sci. USA (in press) 13 S6raphin, B., Boulet, A., Simon, M. and Faye, G. (1987) Proc. Natl Acad. Sci. USA 84, 681045814 14 Chang, S. and Abelson, J. (1987) Genes Dev. 1, 1014-1027 15 Brow, D. and Guthrie, C. (1988) Nature 334, 213-218 16 Schmelzer, C. and Schweyen, R.J. (1982) Nucleic Acids Res. 10, 513-524 17 Jacq, C. etal. (1982)in Mitochondrial Genes (Slonimski, P., Borst, P. and Attardi,G., eds), pp. 155-183, Cold Spring Harbor Press

B~ECHNICALB~IPS

For a number of applications. DNA fragments have to be isolated after agarose gel electrophoresis. However. most methods currently in use are not suitable for the isolation of very large DNA fragments. For purposes such as cloning after pulsed4ield gel electrophor. esist or cloning into yeast artificial chromosomes, intact DNA fragments several hundred kbp in size have to be isolated. For those purposes, as welt as in routine isolations of small DNA pieces, we use the enzyme agarase, which is supplied DNase-free from Calbiocheml,Z The tyophilized enzyme is reconstituted in 50% glycerol at 10 U lalq and can be stored for more than a year at -20oC. The agarose gels are run in low-malting point agarose (BRL). the piece of interest is cut out under long-wave UV light, and equilibrated with 5rnM EDTA and 100 mM salt. The gel is molten at 68oC for 10 minutes, re-equilibrated to 37oC, and enzyme is added (about 2 U per 100 !al gel). Digestion for several hours or overnight completely liquifies the agarose, leaving sugar oligomers. As judged by pulsed-field get electrophoresis, the DNA stays intact after this treatment. It can be used directly for ligations, yeast transformations etc.. or can be carefully ethanol-precipitated after phenol and ether extraction, REFIm~CI~

1 Michiels,E, Burmeistet~M. and Lehrach, H. (1987)Science236, 1305-t308 2 Bucan,M. et aL(1986)EMBOt. 5, 2899-2905 Contributed by Marsft Burme~ed and Hans Lehrach÷, "Department of Pbysiolo~, University of Cal~[ornia, San Franc~o, 513 Parnassus Avenue, San Francisco, CA 94143, USA and +Imperial Cancer Research Fund, PO Box t23, Lincoln'sInn Fields. London WC2A3PX UK

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