The Neurospora Varkud Satellite Ribozyme Abstract the Discovery Of
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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 reverse transcriptase 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 ribozymes. that the Varkud-1c isolate also contained two very abundant RNAs 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 nucleotides 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].