Effects and side-effects of viral RNA silencing suppressors on short RNAs

In eukaryotes, short RNAs play a crucial regulatory role in many processes including development, maintenance of genome stability and antiviral responses. These different but overlapping RNA-guided pathways are collectively termed 'RNA silencing'. To counteract an antiviral RNA silencing response, plant viruses express silencing suppressor proteins. Recent results have shown that silencing suppressors operate by modifying the accumulation and/or activity of short RNAs involved in the antiviral response. Because RNA silencing pathways intersect, silencing suppressors can also inhibit other short-RNA-regulated pathways. Thus, suppressors contribute to viral symptoms. These findings fuel further research to test whether certain symptoms caused by animal viruses are also manifestations of altered RNA regulatory pathways.     

Evidence for a highly nuclease resistant RNA fragment in prosomes

Prosomes, small cytoplasmic particles of mouse erythroblasts were found to contain low molecular weight RNA molecules in the range of 80 nucleotides. Nuclease digestion of prosomes suggests that prosomal proteins cover and protect almost the whole length of their RNA(s). Our results demonstrate clearly that RNA is an intrinsic component of prosomes.     

Completion of RNA synthesis by viral RNA replicases

How the 5′-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA–DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5′-terminus and the C2′-hydroxyl of a ribose at the third nucleotide from the 5′-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.     

Mechanism of RNA silencing by Hfq-binding small RNAs

The stress-induced small RNAs SgrS and RyhB in Escherichia coli form a specific ribonucleoprotein complex with RNAse E and Hfq resulting in translation inhibition, RNAse E-dependent degradation of target mRNAs. Translation inhibition is the primary event for gene silencing and degradation of these small RNAs is coupled with the degradation of target mRNAs. The crucial base-pairs for action of SgrS are confined to the 6 nt region overlapping the Shine-Dalgarno sequence of the target mRNA. Hfq accelerates the rate of duplex formation between SgrS and the target mRNA. Membrane localization of target mRNA contributes to efficient SgrS action by competing with ribosome loading.     

Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation

Cellular micro(mi)RNAs are able to recognize viral RNAs through imperfect micro-homologies. Similar to the miRNA-mediated repression of cellular translation, this recognition is thought to tether the RNAi machinery, in particular Argonaute 2 (AGO2) on viral messengers and eventually to modulate virus replication. Here, we unveil another pathway by which AGO2 can interact with retroviral mRNAs. We show that AGO2 interacts with the retroviral Group Specific Antigen (GAG) core proteins and preferentially binds unspliced RNAs through the RNA packaging sequences without affecting RNA stability or eliciting translation repression. Using RNAi experiments, we provide evidences that these interactions, observed with both the human immunodeficiency virus 1 (HIV-1) and the primate foamy virus 1 (PFV-1), are required for retroviral replication. Taken together, our results place AGO2 at the core of the retroviral life cycle and reveal original AGO2 functions that are not related to miRNAs and translation repression.     

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

Glutaraldehyde nonfixation of isolated viral and yeast RNAs

The RNAs of brome mosaic (BMV), barley stripe mosaic (BSMV), and tobacco mosaic (TMV) viruses were inactivated by reaction with buffered glutaraldehyde. Glutaraldehyde did not fix 4% BMV-RNA, 20% t-RNA, 5% polyadenylic acid, or 5% adenosine monophosphate into water-insoluble precipitates, or gels, in distilled water or in low or high ionic strength buffers nor did it change their ultraviolet (UV) spectra. Two SDS- and phenol-purified commercial yeast RNA preparations from different sources gave UV spectra typical of pure RNA, but could not be freed of a contaminant that reacted with glutaraldehyde by forming a precipitate. The yeast RNAs did not become water-insoluble after glutaraldehyde reaction. BMV-RNA precipitated by Mg2+ could not be cross-linked into an insoluble form by glutaraldehyde. Nonfixation of RNA by glutaraldehyde must be considered in interpretation of attempts to localize RNA by electron microscopy.     

Microarray-based analysis of microbial community RNAs by whole-community RNA amplification

A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Deltafur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.     

Small RNAs in viral infection and host defense

Small RNAs are the key mediators of RNA silencing and related pathways in plants and other eukaryotic organisms. Silencing pathways couple the destruction of double-stranded RNA with the use of the resulting small RNAs to target other nucleic acid molecules that contain the complementary sequence. This discovery has revolutionized our ideas about host defense and genetic regulatory mechanisms in eukaryotes. Small RNAs can direct the degradation of mRNAs and single-stranded viral RNAs, the modification of DNA and histones, and the inhibition of translation. Viruses might even use small RNAs to do some targeting of their own to manipulate host gene expression. This review highlights the current understanding and new insights concerning the roles of small RNAs in virus infection and host defense in plants.     

Microarray-Based Analysis of Microbial Community RNAs by Whole-Community RNA Amplification

A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Δfur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.