Enrichment of midsized RNAs with manganese chloride precipitation.

Enrichment of specific RNAs is important for RNA analysis. MnCl has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl can enrich single-stranded as well as structured RNAs of 450 nt and below from a total RNA preparation.

CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3' Untranslated Region, Altering Polysome Association and Stress Tolerance in .

In , mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3' untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the 3'-UTR probe.

Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host.

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.

The RNA strands of the plus and minus polarities of peach latent mosaic viroid fold into different structures.

It is believed that peach latent mosaic viroid (PLMVd) strands of both the plus and minus polarities fold into similar secondary and tertiary structures. In order to verify this hypothesis, the behavior of both strands in three biophysical assays was examined. PLMVd transcripts of plus and minus polarity were found to exhibit distinct electrophoretic mobility properties under native conditions, to precipitate differently in the presence of lithium chloride, and to possess variable thermal denaturation profiles.

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

Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multibranched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway.

Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA.

Synthesis of wild-type levels of turnip crinkle virus (TCV)-associated satC complementary strands by purified, recombinant TCV RNA-dependent RNA polymerase (RdRp) in vitro was previously determined to require 3' end pairing to the large symmetrical internal loop of a phylogenetically conserved hairpin (H5) located upstream from the hairpin core promoter. However, wild-type satC transcripts, which fold into a single detectable conformation in vitro as determined by temperature-gradient gel electrophoresis, do not contain either the phylogenetically inferred H5 structure or the 3' end/H5 interaction. This implies that conformational changes are required to produce the phylogenetically inferred H5 structure for its pairing with the 3' end, which takes place subsequent to the initial conformation assumed by the RNA and prior to transcription initiation.

A mini-RNA containing the tetraloop, wobble-pair and loop E motifs of the central conserved region of potato spindle tuber viroid is processed into a minicircle.

A Mini-RNA from potato spindle tuber viroid (PSTVd) was constructed specifically for cleavage and ligation to circles in vitro. It contains the C-domain with the so-called central conserved region (CCR) of PSTVd with a 17 nt duplication in the upper strand and hairpin structures at the left and rights ends of the secondary structure. The CCR was previously shown to be essential for processing of in vitro transcripts.

The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo.

The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast.

Chemical mapping of co-existing RNA structures.

In many cases RNA can assume co-existing or meta-stable structures preventing structure determination by chemical mapping. A novel method is described, by which RNA is modified with dimethyl sulphate without shifting the distribution of different structures. The different structures are then separated in native gel electrophoresis, and structure determination by primer extension can be carried out separately for each structure.

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

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.

Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract.

The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular viroids was studied in a nuclear extract from potato suspension cells. The nuclear extract was prepared according to a modified procedure for a plant transcription extract. The transcript of the potato spindle tuber viroid (PSTVd) consists of a monomeric molecule with 17 additional nucleotides, thus doubling most of the central conserved region of viroids of the PSTVd-class.

Inhibition of viroid infection by antisense RNA expression in transgenic plants.

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution.

Structural requirements for viroid processing by RNase T1.

Viroids are replicated via a rolling circle-like mechanism in which (+) strand oligomeric intermediates have to be cleaved enzymatically to unit-length molecules followed by ligation to mature circles. A transcript of potato spindle tuber viroid, which is still infectious, consists of a monomeric molecule with only 22 additional nucleotides, thus doubling part of the central conserved region of viroids. It was shown that this transcript can be cleaved and ligated in vitro to circles by RNase T1.