Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

Abstract

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.

A potential RNA drug target in the hepatitis C virus internal ribosomal entry site

“…The primary sequence of IIId shows only two changes between the six major HCV genotypes, both of which represent covariant substitutions (Fig+ 1B)+ To investigate the functional role of IIId, two mutants were constructed (see Materials and Methods; Fig+ 1)+ The substitution mutants, pIIId264-T-loop and pIIId275CUC, replaced structural elements in the terminal and internal loop, respectively (Fig+ 1B, center and right)+ In vivo analysis indicates that pIIId264-T-loop abrogated IRES activity to 7% of wild-type levels and pIIId275CUC resulted in a reduction to 25%+ Dual substitution mutants within the IIId terminal loop demonstrated reductions in activity in the range of 18-42% for the U264:U269 series (Table 1)+ Single-point mutants within the IIId internal loop at A260 and A276 caused reductions in activity of 12-42% and 45-57%, respectively+ For this series, the mutant A260G produced the most dramatic abrogation of IRES activity for a single substitution mutant+ In ad- The SRL motif, defined as an asymmetric internal loop, (Fig+ 1C), was first identified as a recurrent structural element in 1985 (Branch et al+, 1985)+ The motif is present in both the large subunit rRNA SRL and the loop E region of eukaryotic 5S rRNA, with isosteric sequence variations (Leontis & Westhof, 1998a)+ The structure of the motif, characterized by a series of three non-Watson-Crick base pairs, has been studied extensively using NMR and X-ray crystallography (Wimberly et al+, 1993, Szewczak & Moore, 1995, Correll et al+, 1999)+ The essential structural characteristics of the motif, and the underlying mechanisms leading to its formation have been analyzed systematically (Leontis & Westhof, 1998a)+ As a result, though the motif is defined by conformation rather than by base sequence, the nucleotide requirements for the formation of the motif can be predicted+ Based on this analysis, the motif was predicted to occur twice in the HCV IRES, in Domain IIb and IIId (Fig+ 1A, boxed regions)+ For IIId, this prediction was confirmed by NMR spectroscopy+ Data were acquired on a 27-nt fragment of the IRES, identical in sequence to nt 253-279 (Fig+ 1B, left)+ The distinct SRL geometry leads to a number of nonstandard chemical shift and NOE patterns (Szewczak & Moore, 1995)+ We searched the spectra of IIId for these distinctive patterns, and initiated the assignment process by postulating that they could be identified with the analogous positions within the proposed IIId SRL motif+ Virtually without exception, every diagnostic signal expected for the SRL motif is identified in the IIId fragment (Table 2 and Fig+ 2)+ Figure 2A shows the pattern of NOEs connected with the U259 imino resonance (vertical slice on the right), confirming the formation of a trans-Hoogsteen type U‫ؠ‬A base pair+ The NOE between the U259 imino proton resonance (12+5 ppm) and the A274 H8 resonance (7+4 ppm) is indicative of the reverse Hoogsteen base pairing predicted by the SRL motif+ The NOEs involving the two amino resonances of A274 (centered around 6+5 ppm) are also predicted by the base-pairing scheme+ Moreover, the chemical shift at which the amino resonances appear is itself an indication of the formation of a nonstandard structure+ …”

Simulations of RNA base pairs in a nanodroplet reveal solvation-dependent stability

“…A sheared base pair between adenine and guanine (throughout the text we refer to the nucleotides or nucleosides by the identity of their nitrogenous base) often terminates a tetraloop (5), whereas a reverse Hoogsteen base pair is often observed as part of a loop E or sarcin/ricin loop motif (6,7). The most common and most recognizable are the Watson-Crick base pairs, with 60% of nucleotides in the ribosome being involved in these canonical interactions (8).…”

Structural differences within the loop E motif imply alternative mechanisms of viroid processing

“…A similar role in RNA-RNA interaction has been documented for the loop E motif of the hairpin ribozyme (Butcher and Burke 1994). Several loop E motifs share an exceptional susceptibility to interstrand UV cross-linking involving a specific guanosine and uridine residue (Branch et al 1985).…”

“…Loop E motifs have been identified in both hairpin loops (e.g., the a-sarcin-ricin loop in 23S rRNA; Endo et al 1993) and internal loops like those of bacterial 5S rRNA (Leontis and Westhof 1998a) and Potato spindle tuber viroid (PSTVd) (Branch et al 1985). In bacterial 5S rRNA, the internal loop containing the loop E motif is symmetrical, and the bulged nucleotide characteristic of loop E motifs from eukaryotes and one group of the archaea is missing.…”