The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA frgerprinting and secondary analysis (and additional primer extension studies in the case of the viroid). Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (0) both crosslinks occur in an identical eight-base context, with the sequence 5' GGGAA 3' on one side and the sequence 5' UAC 3' on the other; (it) both crosslinks connect bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as singlestranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules. MATERIALS AND METHODSGel Procedures. Two-dimensional gel electrophoretic analysis (6, 7) was carried out as described (8) (Sankyo, Calbiochem) or pancreatic RNase (Worthington) at 1 mg/ml in the presence of 10 ,g of E. coli tRNA under conditions as described (11,13,14). After digestion, samples were either fractionated by electrophoresis in 20% polyacrylamide gels containing 7 M urea for one-dimensional analysis or were spotted onto cellulose acetate strips as the first step toward the preparation of standard two-dimensional fingerprints (13).Classical Secondary Analysis. Oligonucleotides were recovered either by soaking excised gel bands overnight at 37°C in 100 ,ul of water or by eluting fingerprint spots from DEAE thin-layer plates (13). Secondary enzymatic digestion of oligonucleotides and fractionation of the products were carried out as described (11,13,15 6590The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We analyzed in vivo-labeled RNA to determine which of the two proposed rolling-circle models is more likely to depict the replication cycle of potato spindle tuber viroid. A key feature distinguishing the two models is the presence of a circular monomeric minus strand in one and not the other. Chromatography on cellulose CF1l was used to purify a fraction containing the replication intermediates free from single-stranded progeny. Heat denaturation followed by gel electrophoresis was used to seek possible circular templates-species required for rolling-circle replication to take place. Upon heating, a 32P-labeled RNA was released. Limited nuclease digestion ("nicking") revealed that this was a unitlength circular RNA. Fingerprinting identified it as a plus strand. No circular minus strands were detected in this population or in nuclease-treated samples containing RNase Tl-resistant cores of the replication complex. Thus, potato spindle tuber viroid appears to use an asymmetric pathway in which minus strands are synthesized by rolling-circle copying, but plus strands are not. More details of the replication pathways used by various viroid-like RNAs are needed and will help to establish the evolutionary relationships among these infectious agents.Viroids and related RNAs are the smallest well-defined microorganisms. Interest in the mechanism of viroid replication has broadened recently with the discovery that the 8 agent, the etiologic agent of an often fatal form of hepatitis in man, has a small circular RNA genome (1-3) and may have additional similarities to the small infectious RNAs of plants. A rolling-circle replication cycle was first proposed for viroids in 1981 (4). Although it is now generally accepted that rolling-circle copying occurs during the replication of viroidlike RNAs (5-7), there have been no direct studies to determine which of the two prevailing models more accurately defines the cycle for any particular pathogen. In the original asymmetric model there is a single rolling circle: a circular plus strand* is copied to give a multimeric linear minus strand which directly serves as the plus-strand template (Fig. 1A). In the alternative, symmetric model presented in 1984 (8), the long minus strand is first cleaved to unit length and circularized; it then initiates a second round of rolling-circle copying (Fig. 1B). We identify methods to distinguish these two pathways and, in particular, to ascertain which of these two models accounts for the replication of potato spindle tuber viroid (PSTV).Several lines of evidence suggest that chromatography on cellulose CF11 could be used to prepare the RNAs of the viroid replication complex. For example, this method (9) separates single-stranded RNA (ssRNA) from doublestranded RNA (dsRNA) and has been widely used to purify and analyze the replication intermediates of pathogens which, like viroids (10), have a minus strand composed of RNA. In addition, Northern analysis of RNA gel blots containing control and nuclease-treated samples have shown that ex...
The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.
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