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Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

ABSTRACT: A longer-than-unit-length transcript of potato spindle tuber viroid is correctly processed in a potato nuclear extract only if the central conserved region is folded into a multi-helix junction containing at least one GNRA tetraloop-hairpin. The cleavage-ligation site between G95 and G96 was mapped with S1 nuclease and primer extension. The structural motifs involved in the processing mechanism were analysed by UV crosslinking, chemical mapping, phylogenetic comparison and thermodynamic calculations. For processing, the first cleavage occurs within the stem of the GNRA tetraloop; a local conformational change switches the tetraloop motif into a loop E motif, stabilizing a base-paired 5' end. The second cleavage yields unit-length linear intermediates, whose 3' end is also base-paired and most probably coaxially stacked in optimum juxtaposition to the 5' end. They are ligated to mature circles autocatalytically, with low efficiency, or enzymatically, with high efficiency.

SUBMITTER: Baumstark T

PROVIDER: S-EPMC1169663 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

Baumstark T T Schröder A R AR Riesner D D

The EMBO journal 19970201 3

A longer-than-unit-length transcript of potato spindle tuber viroid is correctly processed in a potato nuclear extract only if the central conserved region is folded into a multi-helix junction containing at least one GNRA tetraloop-hairpin. The cleavage-ligation site between G95 and G96 was mapped with S1 nuclease and primer extension. The structural motifs involved in the processing mechanism were analysed by UV crosslinking, chemical mapping, phylogenetic comparison and thermodynamic calculat ...[more]

PMID: 9034342

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Project description:Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.IMPORTANCE In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

Dataset Information

Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

Publications

Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

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