Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Interaction of RNA with Phospholipid Membranes

RNAs binding with liposomes under near-physiological conditions were obtained by molecular selection. Structural analysis showed that the RNAs could form complexes owing to complementary sequences located in loops. Oligomerization of the RNAs selected was experimentally confirmed. The results and published data testified that formation of high-molecular-weight complexes is a major mechanism increasing the RNA affinity for phospholipid membranes. The role of RNA–membrane interactions in early evolution is discussed in terms of the RNA world hypothesis.     

Leader sequence distinguishes between translatable and encapsidated measles virus RNAs.

The 3'-terminal 55 nucleotides of the negative-strand measles virus RNA genome called the leader sequence is not transcribed into a detectable distinct RNA product. Most of the monocistronic N and bicistronic N-P RNAs lack the leader sequence. However, a subpopulation of the N and N-P RNAs and all of the antigenomes possess this leader. Here, we show that leader-containing subgenomic RNAs are functionally distinct from their leaderless counterparts. In measles virus-infected cells, leaderless monocistronic N and bicistronic N-P RNAs were associated with polysomes. By contrast, leader-containing N and N-P RNAs were found exclusively in nonpolysomal ribonucleoprotein complexes that were resistant to RNase and had a buoyant density of 1.30 g/ml, the same as that of antigenomic ribonucleoprotein complexes. Both antigenomic and subgenomic ribonucleoprotein complexes were specifically immunoprecipitated by antiserum against the N protein, and leaderless RNAs were not found in these complexes. These findings suggest that measles virus distinguishes RNAs destined for encapsidation or translation by the presence or absence of a leader sequence.     

Purification of RNA and RNA-protein complexes by an R17 coat protein affinity method.

We describe an affinity chromatography method to isolate specific RNAs and RNA-protein complexes formed in vivo or in vitro. It exploits the highly selective binding of the coat protein of bacteriophage R17 to a short hairpin in its genomic RNA. RNA containing that hairpin binds to coat protein that has been covalently bound to a solid support. Bound RNA-protein complexes can be eluted with excess R17 recognition sites. Using purified RNA, we demonstrate that binding to immobilized coat protein is highly specific and enables one to separate an RNA of interest from a large excess of other RNAs in a single step. Surprisingly, binding of an RNA containing non-R17 sequences to the support requires two recognition sites in tandem; a single site is insufficient. We determine optimal conditions for purification of specific RNAs by comparing specific binding (retention of RNAs with recognition sites) to non-specific binding (retention of RNAs without recognition sites) over a range of experimental conditions. These results suggest that binding of immobilized coat protein to RNAs containing two sites is cooperative. We illustrate the potential utility of the approach in purifying RNA-protein complexes by demonstrating that a U1 snRNP formed in vivo on an RNA containing tandem recognition sites is selectively retained by the coat protein support.     

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.     

Epigenetic gene regulation by noncoding RNAs

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.     

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.     

Persistent yeast single-stranded RNA viruses exist in vivo as genomic RNA.RNA polymerase complexes in 1:1 stoichiometry

Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins. Both RNAs form ribonucleoprotein complexes with their cognate polymerases. Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles. Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses.     

Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.     

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates the replication of Bamboo mosaic virus and its associated satellite RNA

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3' untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ～35 and ～55 kDa were specifically cross-linked with RNA elements, whereupon the ～35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3' UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3' UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.