Cytoplasmic Z-RNA.

Abstract

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death

“…Alternatively, it is possible that double-stranded vRNA or DI segments may undergo an A-Z transition upon interaction with DAI. Notably, Z-RNA has previously been detected in the cytoplasm and nucleolus of eukaryotic cells (Zarling et al, 1987), and circular dichroism studies have shown that the Zα domain facilitates the transition from A-RNA to Z-RNA under near-physiological conditions (Brown et al, 2000), indicating that dsRNAs in the left-handed Z conformation can serve as physiological substrates for DAI, and for other proteins with Zα domains.…”

Aminoacylation of Synthetic DNAs Corresponding to Escherichia coli Phenylalanine and Lysine tRNAs

“…These interactions enhance the D3'-endo ribose pucker and the anti base conformation typical ofthe RNA A form (23). Thus it is more difficult for an RNA to assume the various polymorphic structures that have been observed for DNA, which is more flexible, although other RNA forms are possible (25). Thus our experiments demonstrating tDNA aminoacylation should not be surprising from a structural perspective, since the absence of the 2' OH in the tDNA must allow the flexibility and polymorphism necessary to achieve the typical RNA solution structure recognized by the appropriate aminoacyl-tRNA synthetase.…”

A mouse chromosome 17 gene encodes a testes-specific transcript with unusual properties

“…It appears that this double helical structure could have Z-RNA-like properties analogous to those observed with Z-DNA (Hall et al 1984). A precedent for the existence of doublestranded cytoplasmic Z-RNA has been reported in protozoa (Zarling et al 1987). The lack of a major open reading frame and the strong potential for secondary structure formation lead to the speculation that the D17Sill testis transcript may serve a structural or regulatory, rather than informational, role during germ cell differentiation.…”