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.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of pol protein

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation

All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.

Dimerization of retroviral RNA genomes : an inseparable pair

Many viruses carry more than one segment of nucleic acid into the virion particle, but retroviruses are the only known group of viruses that contain two identical (or nearly identical) copies of the RNA genome within the virion. These RNA genomes are non-covalently joined together through a process known as genomic RNA dimerization. Uniquely, the RNA dimerization of the retroviral genome is of crucial importance for efficient retroviral replication. In this article, our current understanding of the relationship between retroviral genome conformation, dimerization and replication is reviewed.

Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

The dimerization initiation site (DIS) stem-loop within the HIV-1 RNA genome is vital for the production of infectious virions in T-cell lines but not in primary cells. In comparison to peripheral blood mononuclear cells (PBMCs), which can support the replication of both wild type and HIV-1 DIS RNA mutants, we have found that DIS RNA mutants are up to 100 000-fold less infectious than wild-type HIV-1 in T-cell lines. We have also found that the cell-type-dependent replication of HIV-1 DIS RNA mutants is largely producer cell-dependent, with mutants displaying a greater defect in viral cDNA synthesis when viruses were not derived from PBMCs. While many examples exist of host–pathogen interplays that are mediated via proteins, analogous examples which rely on nucleic acid triggers are limited. Our data provide evidence to illustrate that primary T-lymphocytes rescue, in part, the replication of HIV-1 DIS RNA mutants through mediating the reverse transcription process in a cell-type-dependent manner. Our data also suggest the presence of a host cell factor that acts within the virus producer cells. In addition to providing an example of an RNA-mediated cell-type-dependent block to viral replication, our data also provides evidence which help to resolve the dilemma of how HIV-1 genomes with mismatched DIS sequences can recombine to generate chimeric viral RNA genomes.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process.

An antiviral response directed by PKR phosphorylation of the RNA helicase A

The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.