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Viroids and Virusoids Are Related to Group I Introns

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Viroids and Virusoids Are Related to Group I Introns Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6250-6254, September 1986 Biochemistry Viroids and virusoids are related to group I introns (Tetahymena intervening sequence/potato spindle tuber viroid/mitochondrial conserved sequences/group I introns) GAIL DINTER-GOTTLIEB Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309 Communicated by David M. Prescott, April 28, 1986 ABSTRACT Group I introns are found in nuclear rRNA RNA intron of Tetrahymena thermophila (15-17), Neurospo- genes, mitochondrial mRNA and rRNA genes, and chloroplast ra mitochondrial cob intron 1 (4), and the large ribosomal tRNA genes. The hallmarks of this intron class are a 16- RNA and two messenger RNA introns from yeast mitochon- nucleotide consensus sequence and three sets ofcomplementary dria (18, 19). No enzymes or other proteins are required for sequences. The viroids (circular pathogenic plant RNAs) and these reactions in vitro, the sole requirements being GTP and the virusoids (plant satellite RNAs) also contain the consensus a divalent cation, usually magnesium, and in some cases sequence and the three sets ofcomplementary bases. Pairing of monovalent cations or polyamines. A series of transesterifi- the complementary bases would generate a viroid structure cation reactions occurs, first as guanosine becomes coval- resembling a group I intron, which might be stabilized in vivo ently bonded to the 5' end of the excised intron, then exon through interactions with proteins. The Tetrahymena self- ligation, and the 3' hydroxyl of the intron's 3'-terminal splicing rRNA intron further has sequences- homologous with guanosine attacks a specific bond 4 to 19 bases from the 5' end regions of potato spindle tuber viroid associated with the of the intron, thus creating a covalently closed circle and severity of viroid symptoms. releasing a short oligonucleotide. Both the Tetrahymena and the yeast ribosomal RNA introns cyclize, but no circle form The coding regions of many eukaryotic genes are interrupted has been found for the Neurospora intron. On the basis of by apparently extraneous segments ofDNA, termed introns. similarities in intron structure, it seems reasonable that all In order for the gene to be properly expressed, the introns group I introns splice by a similar mechanism (20). In some must be removed from the RNA transcripts and the residual cases, however, proteins may be necessary for stabilizing the exons must be ligated, a reaction known as RNA splicing. structure and maintaining the base pairing of the boxes. After excision, the intron may exist in a lariat structure (1, 2), Since viroids are single-stranded circular RNAs, similar in as a linear molecule (3-5), or as a circular RNA (5, 6). size to some of the circular introns, comparisons have been One intron class, termed group I, is recognized by con- drawn between the two types of molecules. Diener (21) served sequence and structural features and is found among proposed an intron origin for viroids, and subsequently, mitochondrial messenger and ribosomal RNA genes, noting the similarities between the negative strand of the chloroplast transfer RNA genes, and nuclear ribosomal RNA viroid and the U1 small nuclear ribonucleoprotein particle, genes, a surprisingly broad distribution (3, 7-14). The hall- proposed that the viroids might be escaped introns (22). mark of this intron class is a 16-nucleotide phylogenetically Other homologies with the positive and negative strand conserved sequence (3), the group I consensus sequence viroids were noted by Dickson (23). As additional viroids (Fig. 1 Upper). Twelve bases of this sequence, termed box have been sequenced, further comparisons between the 9L, form a region ofcis-dominant mutations in the splicing of viroids and the various intron types have become possible. the yeast mitochondrial cytochrome b (cob) intron 4. Else- where in the intron is box 2, which is complementary to box RESULTS 9L. Another pair of sequence elements, called box A and box B (or box P and box Q), is less conserved as to sequence but A search of the viroid sequences revealed that the 16- is located upstream of box 9L and is rich in G+C (13). nucleotide group I consensus sequence is present in all ofthe Although no splicing-defective mutants have yet been found viroids.* In fact, this sequence represents the lower portion in vivo, mutations introduced into this region in the of the "central conserved region" of the viroids (24). This Tetrahymena intervening sequence cause defective splicing region is centrally located when the structure is written in the ofthe RNA transcript (13). A third pair of sequences, box 9R canonical rod form (ref. 25; Fig. 2 Upper). As shown in Fig. and box 9R', although not conserved as to sequence, are 2 Lower, the sequence is present even in viroids such as conserved in location. Box 9R is locatedjust downstream of CCCV, which has only 11% sequence homology with the 9L, and 9R' is just upstream of A. The ability of 9R and 9R' PSTV group (24). Surprisingly, it is found in virusoids as well. to base pair has been implicated in yeast cob mRNA splicing, Virusoids are covalently closed, single-stranded, circular since a point mutation at the base of the helix destroys RNA molecules of 300-400 nucleotides, which are found splicing, while a double mutation, restoring pairing, also encapsidated with other, much larger, single-stranded, linear restores splicing of the intron (14). RNAs in some viruses. The RNA of such a virus is termed The pairing of the complementary boxes, and the order in RNA 1, while the accompanying satellite RNA is RNA 2. which they occur within the intron, 9R', A, B, 9L, 9R, and Two of the virusoids that have been sequenced, velvet 2, force the RNA into a characteristic structure (Fig. 1 tobacco mottle virus RNA 2 (= VTMoV) and Solanum Upper). In this case, the box 9L-box 2 pairing would be at the level of tertiary structure. Abbreviations: PSTV, potato spindle tuber viroid; CCCV, coconut The phenomenon of self-splicing in vitro has been de- cadang-cadang viroid; CEV, citrus exocortis viroid; TASV, tomato scribed for several group I introns, the nuclear ribosomal apical stunt viroid; TPMV, tomato planta macho viroid; VTMoV, velvet tobacco mottle virusoid; SNMV, Solanum nodiflorum mottle virusoid. The publication costs of this article were defrayed in part by page charge *Dinter-Gottlieb, G. & Cech, T. R. (1984) in Molecular Basis of payment. This article must therefore be hereby marked "advertisement" Plant Disease Poster Abstracts, eds. Kosuge, T. & Timberlake, W. in accordance with 18 U.S.C. §1734 solely to indicate this fact. (Univ. of California, Davis, CA), p. 10. 6250 Downloaded by guest on September 28, 2021 Biochemistry: Dinter-Gottlieb Proc. Natl. Acad. Sci. USA 83 (1986) 6251 GROUP I CONSENSUS Box U & G U{AU U GC UA U U UG UU AUAUGGAU UG c \ III II CII I A U UAGACAACUGG G AAC A- U U *D\\* G-C BOX2 UC G A G G GC A U A AG U \ U A A AFUAC\\GGGU\ \ UGA G A AGC GG G A IC U CAU AACAU A 5 16N Ac G c A A GG U GGUUUAAAGGC C G G U CG CC AC AC A \\ ~~~U I1 U GA GAG ACGAC\\\CCGACA A ACCAAAUUUCUGA A G AUA AG \\ACU GG UU //A G G /U AAU GUA \\\GA GU A AuC 3-C U- G-U G- C G-C UA A- U A U G 3-C 3-U G 31 A A C G u~uA U-A G-cG i-A G-C CCt U- A-U D STEM U-A A- U A G A CG U U C U A A-A UC UA A C U C U U G C C U U U U-G C U-G U-G C-G G-C A C C-G GROUPI CONSENSUS G A G C-G C-G C-G U U BOX 2 C-G G-U /GG- ic~ C U U A-U L C A-U C U U U K 9R G A G C c GS CU-G C CC UUG GA A C CG CA GGAACUAAA C AAA IIIII IIIII 1H il l11 C UCCACCUUGGUGU CCUUGGU U GG G GA A AAGA AG U G~~A A U C C A CC C\ A A GG cu G C U C\CC C GUI//C CU A A U uC A G A G GG C U A G G G>~A C AC C G ,U A /GC G G //UG G 'U UUCG / A G7 'uCP/C G AA AA G a GGA ~~CA STE~M" FIG. 1. (Upper) Structure of a group I intron (exemplified by the self-splicing intron of Tetrahymena) generated by pairing box 9R with box 9R' and box A with box B in the secondary structure, and box 9L with box 2 at the level of tertiary structure [after Michel and Dujon (7)]. On the right, 16N indicates 16 nucleotides not shown. (Lower) Potato spindle tuber viroid (PSTV) structure derived from pairing the conserved sequence elements that are also found in group I introns. nodiflorum mottle virus RNA 2 (= SNMV) (26) contain the paired, a structure is generated in the viroid which is group I consensus sequence and box sequences (Fig. 2 strikingly similar to that of group I introns in the region of the Lower), although the virusoids have little sequence homology boxes (Fig. 1 Lower). The calculated free energy for folding with the viroids. ofthis molecule, approximately -100 kcal/mol (1 cal = 4.184 The conserved sequence elements of group I introns also J), is not nearly as favorable as that calculated for the rod occur in the viroids.* Box 9L, a portion of the consensus structure, -209.8 kcal/mol (M.
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