Enrichment of midsized RNAs with manganese chloride precipitation.

Enrichment of specific RNAs is important for RNA analysis. MnCl2 has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl2 can enrich single-stranded as well as structured RNAs of 450 nt and below from a total RNA preparation. We have applied this method to map the transcription start sites of a PSTVd transcript from total RNA from yeast under conditions where the RNA was previously undetectable.

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

In Cryptococcus neoformans, 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 RPL2 3'-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C. neoformans and also bound the 3'-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3' UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis.IMPORTANCE Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.

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. 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.

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. Subsequently, the structure of PLMVd transcripts of minus polarity was elucidated by biochemical methods, thereby permitting comparison to the known structure of the plus polarity. Specifically, enzymatic probing, electrophoretic mobility shift assay, and ribonuclease H hydrolysis were performed in order to resolve the secondary structure of the minus polarity. The left domains of the strands of both polarities appear to be similar, while the right domain exhibited several differences even though they both adopted a branched structure. The pseudoknot P8 formed in the plus strand seemed not formed in the minus strands. The structural differences between the two polarities might have important implications in various steps of the PLMVd life cycle.

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. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson-Crick GC pairs results in a significant overall stabilization of the CVd-III loop E-like motif. Unlike PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5' side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3' sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5' and 3' termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5' and 3' termini of CVd-III in position for the host ligase.

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. The DR region, located 140 nucleotides upstream from the 3' end and previously determined to be important for transcription in vitro and replication in vivo, is proposed to have a role in the conformational switch, since stabilizing the phylogenetically inferred H5 structure decreases the negative effects of a DR mutation in vivo. In addition, high levels of aberrant transcription correlate with a specific conformational change in the Pr while maintaining the same conformation of the 3' terminus. These results suggest that a series of events that promote conformational changes is needed to expose the 3' terminus to the RdRp for accurate synthesis of wild-type levels of complementary strands in vitro.