Characterisation and comparison of bovine RNA polymerase III promoters for RNA interference

The characterisation of novel bovine RNA polymerase III promoters for the expression of short hairpin RNAs for gene silencing resulted in the identification of a highly efficient promoter sequence. The replication of bovine viral diarrhea virus was them suppressed using short hairpin RNAs expressed form this promoter in vitro.

Discovery of structural and functional features in RNA pseudoknots

An RNA pseudoknot consists of nonnested double-stranded stems connected by single-stranded loops. There is increasing recognition that RNA pseudoknots are one of the most prevalent RNA structures and fulfill a diverse set of biological roles within cells, and there is an expanding rate of studies into RNA pseudoknotted structures as well as increasing allocation of function. These not only produce valuable structural data but also facilitate an understanding of structural and functional characteristics in RNA molecules. PseudoBase is a database providing structural, functional, and sequence data related to RNA pseudoknots. To capture the features of RNA pseudoknots, we present a novel framework using quantitative association rule mining to analyze the pseudoknot data. The derived rules are classified into specified association groups regarding structure, function, and category of RNA pseudoknots. The discovered association rules assist biologists in filtering out significant knowledge of structure-function and structure-category relationships. A brief biological interpretation to the relationships is presented, and their potential correlations with each other are highlighted.

Viral RNA silencing suppressors (RSS) : novel strategy of viruses to ablate the host RNA interference (RNAi) defense system

Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host–virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.

Mining characteristic relations bind to RNA secondary structures

The identification of RNA secondary structures has been among the most exciting recent developments in biology and medical science. It has been recognized that there is an abundance of functional structures with frameshifting, regulation of translation, and splicing functions. However, the inherent signal for secondary structures is weak and generally not straightforward due to complex interleaving substrings. This makes it difficult to explore their potential functions from various structure data. Our approach, based on a collection of predicted RNA secondary structures, allows us to efficiently capture interesting characteristic relations in RNA and bring out the top-ranked rules for specified association groups. Our results not only point to a number of interesting associations and include a brief biological interpretation to them. It assists biologists in sorting out the most significant characteristic structure patterns and predicting structurefunction relationships in RNA.

Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation

Cordycepin (3′ deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3′ hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3′ end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3′ end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Replacement of RNA hairpins by in vitro selected tetranucleotides

An in vitro selection method based on the autolytic cleavage of yeast tRNAPhe by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucieotide sequences allow proper folding of the rest of the tRNA molecule, wheras others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNAPhe and the anticodon hairpin of E.coli tRNAPhe without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally non essential hairpins in any RNA.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted -propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

A role for SSU72 in balancing RNA polymerase II transcription elongation and termination

Interactions of pre-mRNA 3′end factors and the CTD of RNA polymerase II (RNAP II) are required for transcription termination and 3′end processing. Here, we demonstrate that Ssu72p is stably associated with yeast cleavage and polyadenylation factor CPF and provide evidence that it bridges the CPF subunits Pta1p and Ydh1p/Cft2p, the general transcription factor TFIIB, and RNAP II via Rpb2p. Analyses of ssu72-2 mutant cells in the absence and presence of the nuclear exosome component Rrp6p revealed defects in RNAP II transcription elongation and termination. 6-azauracil, that reduces transcription elongation rates, suppressed the ssu72-2 growth defect at 33°C. The sum of our analyses suggests a negative influence of Ssu72p on RNAP II during transcription that affects the commitment to either elongation or termination.

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.