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12 Steitz, T. A. and Steitz, J. A. (1993) Proc. Natl. Acad. 22 Burgin, Jr, A. B., Gonzalez, C., Matulic-Adamic, J., Karpeisky, Sci. U.S.A. 90, 6498–6502 A. M., Usman, N., McSwiggen, J. A. and Beigelman, L. 13 Pontius, B. W., Lott, W. B. and von Hippel, P. H. (1997) (1996) Biochemistry 35, 14090–14097 Proc. Natl. Acad. Sci. U.S.A. 94, 2290–2294 23 Clouet-d’Orval, B. and Uhlenbeck, O. C. (1997) 14 Zhou, D.-M. and Taira, K. (1998) Chem. Rev. 98, Biochemistry 36, 9087–9092 991–1026 24 Murray, J. B. and Scott, W. G. (2000) J. Mol. Biol. 296, 15 Scott, W. G. and Klug, A. (1996) Trends Biochem. Sci. 21, 33–41 220–224 25 Blount, K. F., Grover, N. L., Molker, V., Beigelman, L. and 16 Scott, E. C. and Uhlenbeck, O. C. (1999) Nucleic Acids Uhlenbeck, O. C. (2002) Chem. Biol. 9, 1009–1016 Res. 27, 479–484 26 Cohen, S. B. and Cech, T. R. (1997) J. Am. Chem. Soc. 17 Peracchi, A., Beigelman, L., Scott, E. C., Uhlenbeck, O. C. 119, 6259–6268 and Herschlag, D. (1997) J. Biol. Chem. 272, 26822–26826 27 Blount, K. F. and Uhlenbeck, O. C. (2002) Biochemistry 41, 18 Wang, S., Karbstein, K., Peracchi, A., Beigelman, L. and 6834–6841 Herschlag, D. (1999) Biochemistry 38, 14363–14378 28 Stage-Zimmermann, T. K. and Uhlenbeck, O. C. (2001) 19 Murray, J. B., Seyhan, A. A., Walter, N. G., Burke, J. M. and Nat. Struct. Biol. 8, 863–867 Scott, W. G. (1998) Chem. Biol. 5, 587–595 20 O’Rear, J. L., Wang, S., Feig, A. L., Beigelman, L., Uhlenbeck, O. C. and Herschlag, D. (2001) RNA 7, 537–545 21 Bondensgaard, K., Mollova, E. T. and Pardi, A. (2002) Biochemistry 39, 11532–11542 Received 27 August 2002

The Neurospora Varkud satellite R. A. Collins1 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Abstract isolates contained plasmid that were not Presented is a review of the discovery and charac- derived from the mt chromosome [1,2]. The best terization of the Neurospora Varkud satellite characterized of these plasmids is the Mauriceville ribozyme. It outlines the approaches and obser- plasmid, which encodes a vations that have led to our current level of (RT) that selectively makes cDNA copies of its understanding of the structure and function of this own mRNA [3]. ribozyme, and highlights its distinctive features In addition to containing several novel introns compared with other naturally occurring small and a plasmid of the Mauriceville type, we noticed . that the Varkud-1c isolate also contained two very abundant of approx. 0.9 kb, as de- duced from electrophoretic mobility studies on The discovery of Varkud satellite (VS) agarose gels. The RNAs are so abundant that they RNA were easily detectable by ethidium bromide stain- ing of total mt RNA, suggesting that these novel The discovery of the VS ribozyme was a classic RNAs are present at concentrations similar to case of serendipity that developed from our pre- those of the mt rRNAs and tRNAs, and much vious studies of the mitochondrial (mt) genomes higher than the concentration of any mRNA or of natural isolates of Neurospora. As is typical of excised Group I intron RNA. The Varkud-1c iso- fungi and other simple eukaryotes, the standard late also contains a Group II intron which was also laboratory strains of Neurospora contain Group I present at concentrations detectable by ethidium introns in several of their mt genes. We, and bromide staining (because of their covalently others, had previously found that many natural closed ‘lariat’ structure, Group II introns are isolates of Neurospora contained several introns thought to be less susceptible to degradation not found in the laboratory strains, and some than linear RNAs). So, our initial hypothesis was that the novel 0.9 kb RNAs might be previously unrecognized, stable, excised Group II intron Key words: mitochondrial plasmid, RNA structure, Varkud satellite RNAs. RNA. To identify the genes on the mt chromosome Abbreviations used: HDV, hepatitis delta virus; mt, mitochondrial; RT, reverse transcriptase; VS, Varkud satellite. which encoded these putative introns, we excised 1E-mail rick.collins!utoronto.ca the 0.9 kb RNAs from a gel, and made radioactive

# 2002 Biochemical Society 1122 Ribozymes and RNA Catalysis

probes using random hexamer primers, labelled 5h hydroxy termini, the same as those produced dNTPs and RT. These probes were hybridized to by the hammerhead, hairpin, and hepatitis delta Southern blots of restriction-digested Varkud-1c virus (HDV) ribozymes [4]. mtDNA (whose restriction map had been deter- The majority of VS RNA isolated from mined previously). Rather than hybridizing to mitochondria is in the form of monomers, identical the mt chromosome, the probes hybridized to a in sequence to one repeat unit of the multimeric ladder of bands typical of the pattern produced by VS DNA plasmid. Most of these RNAs are hybridization to a multimeric plasmid, a pattern circular, with smaller amounts of linear monomers which was immediately recognizable to us because and multimeric forms also detected. These obser- of our previous work with other mt plasmids. vations, and analogies with previously charac- Further experiments showed that the two 0.9 kb terized ribozymes, suggested that the role of RNAs were actually circular and linear forms of the VS ribozyme is to process multimeric tran- the RNA transcribed from this novel plasmid. scripts into monomeric form by site-specific self- The plasmid was named VS [4], referring to the cleavage. Indeed, processing of multimeric VS Varkud isolate in which it was found, and to the RNAs into all of the expected cleavage and ligation observation that it is found only in strains that also products was observed in reactions that contained contain the RT-encoding plasmid [5], suggesting only RNA, buffer, salt and MgCl#, indicating that that it is a satellite of the RT-encoding plasmid, in the VS ribozyme was capable of ligation as well as the same sense of the word as used by plant cleavage [7]. The absolute and relative efficiency virologists to describe an RNA that depends on of cleavage and ligation varies substantially among another DNA or RNA of unrelated sequence for versions of VS RNA that contain different subsets its replication, propagation and\or maintenance. of the native VS sequence [8–10]. As a generaliz- We cloned and sequenced the VS plasmid ation, ligation is easily observable using constructs DNA, and found no open reading frames of and\or reaction conditions in which the two prod- significant length, nor did BLAST searches reveal ucts of self-cleavage remain associated with each similarity to any other sequence in GenBank2.We other [7,10–12]. have identifed very closely related versions of the VS plasmid in several other Neurospora isolates, but the evolutionary origin of VS remains un- Characterization and properties of known. The VS plasmid DNA contains a sequence the VS ribozyme that matches the consensus for the minimal pro- Preparation of uncleaved precursor RNA for kin- moter on the Neurospora mt genome, and VS can etic analysis of the cleavage reaction was achieved be transcribed in mt extracts by the mt RNA by in vitro transcription in the presence of a polymerase which initiates transcription at the decreased concentration of Mg#+ [13]. We later site expected for a mt transcript [6]. realized that lower Mg#+ concentrations also re- The discovery of the catalytic activity of VS duced the tendency of T7 RNA polymerase to add RNA came about completely by accident. We had non-templated to the 3h end of RNA cloned the VS plasmid DNA into vectors that transcripts: the ability to synthesize a population contained a T7 promoter, for the purpose of of RNAs, nearly all of which had the same 3h end, synthesizing strand-specific probes by in vitro greatly increased the quality of chemical and transcription to be used in Northern hybridization enzymic modification data [11]. The VS ribo- experiments to determine which strand of the zyme is active in the presence of millimolar plasmid DNA was transcribed. When we checked concentrations of several bivalent cations, with the quality of the probes by gel electrophoresis and Mg#+ being the most effective [13]. Molar concen- autoradiography we found that both contained trations of certain univalent cations also support RNAs of the expected length, but two smaller activity, although at lower rates, with Li+ being bands were also present in one of the samples. The the most effective ([14]; J. E. Olive and R. A. sum of the sizes of the two smaller bands was equal Collins, unpublished work). In certain reaction to that of the expected full-length RNA, suggesting conditions, spermine or the cationic peptide anti- that they might be products of cleavage at a specific biotic viomycin alter the Mg#+-dependent reaction site. Additional experiments confirmed that RNA- to favour trans-cleavage reactions, even with VS catalysed site-specific cleavage did occur in the T7 RNAs that normally cleave themselves (cis- transcription mixture, and that the cleavage cleavage) [15,16]. Cobalt hexammine, an analogue products contained 2h,3h-cyclic phosphate and of hexahydrated Mg#+ that supports catalysis of the

1123 # 2002 Biochemical Society Biochemical Society Transactions (2002) Volume 30, part 6

hairpin ribozyme, does not support VS activity, RNA. When we later deduced the secondary although it does lead to almost correct folding (as structure of the VS ribozyme (Figure 1a) [18], we inferred from chemical modification protection were surprised to find that the most efficient trans- experiments), and it co-operates, rather than cleaving system, derived from DNA fragments competes, with Mg#+ in facilitating cleavage [17]. subcloned at the AvaI restriction site, consisted of The minimal contiguous portion of VS RNA a substrate that corresponded to most of stem-loop required for ribozyme activity was identified by I of the cis-cleaving RNA and a ribozyme (called the small amount of comparative sequence data, the Ava ribozyme) that corresponded to helices linker insertion mutagenesis and deletion muta- II–VI [19]. Unlike trans-cleaving versions of the genesis [8]. During the course of these exper- hammerhead, hairpin or HDV ribozymes, the VS iments, we also divided the VS plasmid DNA ribozyme does not use long stretches of com- clones into two pieces by restriction plementary base pairing to associate with its sub- digestion at each of several unique restriction sites strate; nonetheless, the ribozyme–substrate inter- that occurred in this region. Separate RNAs were action is quite tight, inferred from apparent Km transcribed from each of the cloned subfragments, values of trans-cleavage reactions that are in the and combined pairwise to identify trans-acting range of 0.2 to 1 µM [19,20]. Also, the VS versions of the VS ribozyme that could bind and ribozyme has no specific sequence requirements cleave the target site even when it was on a separate upstream of the cleavage site [8]; this feature has allowed it to be adapted as a tool to produce a homogeneous 3h end on any RNA of interest made by in vitro transcription [21]. Our laboratory and Figure 1 others have used the Ava ribozyme version of VS, Models of the secondary and tertiary structure of the or its derivatives, in a variety of kinetic and folding VS ribozyme studies that have identified bases, interactions, Helices are numbered with roman numerals and nucleotides are and structures that contribute to the activity of the numbered according to [4]. The cleavage site is indicated by the VS ribozyme [19,20,22,23]. arrowhead in parts (a) and (b); the cleavage site is on the back of stem-loop I in part (c). See text for more details.

Secondary and tertiary structure Secondary structure models of VS were developed by attempting to maximize the correlation be- tween chemical modification structure probing data and MFOLD predictions of possible RNA secondary structures. The relevance of models to the function of the ribozyme was tested by making mutations that were predicted to disrupt base- paired regions and making compensatory substi- tutions that were predicted to restore pairing, but with different base pairs. This approach produced a consistent model for stem-loops II–VI [18], but the functional structure of stem-loop I, which contains the site of self-cleavage in an internal loop, remained enigmatic until in vitro selection experiments and subsequent site-directed muta- genesis revealed that stem-loop I undergoes a substantial change in its base pairing during Mg#+- dependent folding with the rest of the ribozyme [10]. This conformational change in stem-loop I, to form the ‘shifted’ conformation (indicated in Figure 1a), is facilitated by a ‘kissing’ interaction (pseudoknot) with nucleotides in the hairpin loop of stem-loop V. Indeed, stem-loop V alone is sufficient to induce the shifted conformation of

# 2002 Biochemical Society 1124 Ribozymes and RNA Catalysis

stem-loop I [24]. NMR structures have recently paired helix eliminates activity [30]. Surprisingly, been determined for the internal loop that con- certain other structural motifs can replace the 730 tains the cleavage site in either the shifted loop and provide site-specific cleavage activity (B. Hoffmann, G. Mitchell, A. A. Andersen, R. A. that is up to four orders of magnitude above the Collins and P. Legault, unpublished work) or background rate of uncatalysed breakage of the unshifted [25,26] conformations, and provides phosphodiester backbone [30]. So, it seems that evidence that a metal-ion binding motif that is the region of the RNA occupied by the 730 loop is important for activity and is predicted to form important for activity, but its exact role is still only in the shifted conformation [10] does indeed unknown. form. Binding experiments have shown that mu- Subsequent interference experiments have tants of stem-loop I that constitutively adopt the identified additional atoms that are important for shifted conformation bind more tightly to the activity and which map to the proposed core of the ribozyme than does wild-type stem-loop I, and ribozyme [31]. The functional substitution of a much more tightly than mutants that cannot adopt uridine-turn in the III–IV–V junction with helices the shifted conformation (R. Zamel and R. Collins, of various lengths suggests that this three-way unpublished work). The kissing interaction may junction may resemble a four-way junction (with also help to position stem-loop I, and hence the the uridine-turn as the fourth ‘helix’) and further cleavage site, in the appropriate orientation with supports the proposed orientation of the helices regard to the rest of the ribozyme to facilitate [32]. Lilley and co-workers have recently used catalysis. fluorescence resonance energy transfer and gel In 1998, we proposed a model of the approxi- electrophoretic mobility to study RNA molecules mate relative orientations of the helices in VS that contained VS junction sequences and artificial (Figure 1b) [9,30]. This model accommodated: (i) helices lacking internal loops and bulged nucleo- the kissing interaction between bases in loops I tides [22,23]. They have proposed a model for the and V, which showed that these loops must be near preferred helix orientations induced by the folding each other in the functional structure [27]; (ii) a of these junctions, which is in general agreement short-wavelength UV cross-link between A652 with previous models, suggesting that the helix and A761 that formed only in the presence of junctions play a significant role in directing the Mg#+, which suggested that these regions of helices global folding of the RNA. UV cross-linking of II and VI were also in close proximity (D. 4-thio-uridine incorporated adjacent to the site DeAbreu, T. Mittermaier and R. Collins, unpub- of cleavage showed that this cross-links lished work); (iii) site-directed deletion muta- with high efficiency to A756 in the 730 loop in tions that had been designed to shorten helices helix VI only under conditions that support predicted by the secondary structure mode [9]; catalytic activity, adding further refinement to the and (iv) data from nucleotide analogue and chemi- model and providing the first physical evidence for cal modification interference and protection ex- the location of the cleavage site in relation to the periments [18,28,29] that suggested a core region other helices. Hydroxyl radical protection exper- that contained the nucleotides that were important iments identified regions of the RNA that are for activity. This model raised the possibility that protected from solvent (and therefore on the the cleavage site might be in close proximity to ‘inside’) when the RNA is folded in the presence what is now called the 730 loop in helix VI and to of Mg#+. Additional experiments with mutants in part of helix II, and that these regions of the which stem-loop I is either deleted or prevented ribozyme could play a role in the chemical step of from interacting efficiently with the rest of the the reaction [29]. Direct evidence that the active ribozyme (due to disruption of the kissing inter- site is contained within helices I, II, VI and the action with loop V) revealed a cluster of nucleo- lower (as drawn in Figure 1a) portion of III was tides that may identify the binding surface obtained by deleting helices IV, V and the upper between stem-loop I and the rest of the ribozyme portion of III in a stem-loop I mutant that [33]. A working model for the three-dimensional constitutively adopts the shifted conformation, orientation of the helices in VS is shown in Fig- and is therefore not dependent on the kissing ure 1(c). Detailed models will need to accom- interaction with loop V [30]. Mutational analyses modate the constraints implied by the hydroxyl have revealed that substitution of individual bases radical protection data, and other biochemical and in the 730 loop substantially decreases activity mutational data that bear on the functional struc- [20,30], and conversion of the 730 loop into a base- ture of the ribozyme.

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References 17 Maguire, J. L. and Collins, R. A. (2001) J. Mol. Biol. 309, 45–56 1 Collins, R. A. and Lambowitz, A. M. L. (1988) Plasmid 9, 18 Beattie, T. L., Olive, J. E. and Collins, R. A. (1995) 53–70 Proc. Natl. Acad. Sci. U.S.A. 92, 4686–4690 2 Griffiths, A. J. F. (1995) Microbiol. Rev. 59, 673–685 19 Guo, H. C. and Collins, R. A. (1995) EMBO J. 14, 368–376 3 Kuiper, M. and Lambowitz, A. M. L. (1988) Cell 56, 20 Lafontaine, D. A., Wilson, T. J., Norman, D. G. and Lilley, 693–704 D. M. (2001) J. Mol. Biol. 312, 663–674 4 Saville, B. J. and Collins, R. A. (1990) Cell 61, 685–696 21 Ferre! -d’Amare, A. R. and Doudna, J. A. (1996) Nucleic 5 Collins, R. A. and Saville, B. J. (1990) Nature (London) 345, Acids Res. 24, 977–978 177–179 22 Lafontaine, D. A., Norman, D. G. and Lilley, D. M. (2001) 6 Kennell, J. C., Saville, B. J., Mohr, S., Kuiper, M. T., Sabourin, EMBO J. 20, 1415–1424 J. R., Collins, R. A. and Lambowitz, A. M. (1995) 23 Lafontaine, D. A., Norman, D. G. and Lilley, D. M. (2002) Genes Dev. 9, 294–303 EMBO J. 21, 2461–2471 7 Saville, B. J. and Collins, R. A. (1991) Proc. Natl. Acad. 24 Andersen, A. A. and Collins, R. A. (2001) Proc. Natl. Acad. Sci. U.S.A. 88, 8826–8830 Sci. U.S.A. 98, 7730–7735 8 Guo, H. C., De Abreu, D. M., Tillier, E. R., Saville, B. J., 25 Flinders, J. and Dieckmann, T. (2001) J. Mol. Biol. 308, Olive, J. E. and Collins, R. A. (1993) J. Mol. Biol. 232, 665–679 351–361 26 Michiels, P. J., Schouten, C. H., Hilbers, C. W. and Heus, 9 Rastogi, T. and Collins, R. A. (1998) J. Mol. Biol. 277, H. A. (2000) RNA 6, 1821–1832 215–224 27 Rastogi, T., Beattie, T. L., Olive, J. E. and Collins, R. A. 10 Andersen, A. A. and Collins, R. A. (2000) Mol. Cell 5, (1996) EMBO J. 15, 2820–2825 469–478 28 Beattie, T. L. and Collins, R. A. (1997) J. Mol. Biol. 267, 11 Hiley, S. L., Sood, V. D., Fan, J. and Collins, R. A. (2002) 830–840 EMBO J. 21, 4691–4698 29 Sood, V. D., Beattie, T. L. and Collins, R. A. (1998) 12 Jones, F. D., Ryder, S. P. and Strobel, S. A. (2001) Nucleic J. Mol. Biol. 282, 741–750 Acids Res. 29, 5115–5120 30 Sood, V. D. and Collins, R. A. (2002) J. Mol. Biol. 320, 13 Collins, R. A. and Olive, J. E. (1993) Biochemistry 32, 443–454 2795–2799 31 Sood, V. D., Yekta, S. and Collins, R. A. (2002) Nucleic 14 Murray, J. B., Seyhan, A. A., Walter, N. G., Burke, J. M. and Acids Res. 30, 1132–1138 Scott, W. G. (1998) Chem. Biol. 5, 587–595 32 Sood, V. D. and Collins, R. A. (2001) J. Mol. Biol. 313, 15 Olive, J. E., De Abreu, D. M., Rastogi, T., Andersen, A. A., 1013–1019 Mittermaier, A. K., Beattie, T. L. and Collins, R. A. (1995) 33 Hiley, S. L. and Collins, R. A. (2001) EMBO J. 20, EMBO J. 14, 3247–3251 5461–5469 16 Olive, J. E. and Collins, R. A. (1998) Biochemistry 37, 6476–6484 Received 27 August 2002 The Ribosome

Biochemical identification of A-minor motifs within RNA tertiary structure by interference analysis S. A. Strobel1 Department of Molecular Biophysics and Biochemistry, Department of Chemistry, 260 Whitney Avenue, Yale University, New Haven, CT 06520-8114, U.S.A.

Abstract chemical identification of these interactions is A-minor motifs are the most common tertiary now feasible using interference mapping analysis structural elements in RNA helix packing. Bio- with the adenosine analogues 2h-deoxyadenosine and 3-deaza-adenosine. This approach was used to demonstrate that A-minor motifs mediate helix packing interactions that are important for 5h- Key words: group I intron, nucleotide analogue interference splice site selection in the group I intron. By mapping (NAIM), ribozyme. Abbreviations used: c3AαS, 5h-O-(1-thio)-3-deaza-adenosine tri- analysing the interference pattern of several ana- phosphate; dAαS, 2h-deoxyadenosine; NAIM, nucleotide ana- logues it is possible to identify and distinguish logue interference mapping; NAIS, nucleotide analogue inter- the four variants of the A-minor motif. ference suppression. 1To whom correspondence should be addressed at the present Introduction address: Creighton University, Department of Chemistry, 2500 California Plaza, Omaha, NE 68178, U.S.A. (e-mail strobel! The family of A-minor motifs appears to be the mail.csb.yale.edu). most ubiquitous helix-packing elements within

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