A Novel Algorithm for Identification of Activated Cryptic 5′ Splice Sites

Abstract

The resonances of the protonated carbons of [d(TAGCGCTA)]₂ have been assigned by the two-dimensional proton-detected double-quantum heteronuclear correlation experiment ([¹H-^l3C]-DQCOSY). ¹³C-coupled and ^l3C-decoupled versions of the experiment were used. The assignment method is discussed in detail. The deoxyribose cross peaks segregate into five well-resolved regions, and the base cross peaks have distinct features that are helpful for assignments. The cross peaks from the ¹H-¹³C pairs at the Cyd5, Ado2 and ThdCH₃ base positions fall in separate regions of the spectrum from each other; they also are resolved from the closely spaced Ado8, Guo8, Cyd6 and Thd6. Additional parameters for distinction of the base signals are their differing J-coupling values and long-range coupling patterns.     

A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.     

RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1α in Supporting Brucella Replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α^−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

A Genome-Scale RNA–Interference Screen Identifies RRAS Signaling as a Pathologic Feature of Huntington's Disease

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington''s disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington''s disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington''s disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington''s disease.     

A New Internal Ribosomal Entry Site 5′ Boundary Is Required for Poliovirus Translation Initiation in a Mouse System

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1–8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.The single-stranded genome of poliovirus has mRNA polarity, is approximately 7,500 nucleotides (nt) in length, is polyadenylylated (45), and is linked covalently at its 5′ end to a small protein called VPg (30, 41). The RNA itself is infectious; cells transfected with the RNA produce progeny virions that are infectious. Poliovirus RNA harbors a long 5′ noncoding region of approximately 740 nt that is important for viral RNA and protein syntheses. A possible cloverleaf-like structure formed by the 5′-proximal end of the RNA (approximately 90 nt) is a probable cis element that regulates the synthesis of the plus-strand RNA (1). nt 120 to 590 of the poliovirus RNA make up the internal ribosomal entry site (IRES) (32), which directs the viral translation initiation step in a 5′-end- and cap-independent manner (17, 25, 29, 44). The IRES is assumed to carry a number of secondary structures (10, 40), and multiple host cellular factors are required for its functions.Translation of poliovirus does not occur in a cell-free wheat germ translation system, and it occurs only inefficiently and usually incorrectly in rabbit reticulocyte lysates (RRL) (9). The poor translation in RRL, however, is markedly improved by the addition of factors from HeLa cells (5, 9, 33). Other IRESs, such as the IRESs of encephalomyocarditis virus RNA (18) and hepatitis C virus RNA (43), are highly functional in the RRL system. These observations indicate that individual IRESs with different structures may require quantitatively and/or qualitatively different sets of host factors for their activities.Determinants for strain-specific neurovirulence (replication ability) of poliovirus type 1 in the central nervous system (CNS) have been mapped in the IRES region, particularly at nt 480 of the genome, by using monkey neurovirulence tests on recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains (19, 24). Similar results were obtained when the recombinants were tested for their relative neurovirulence levels by using transgenic (Tg) mice carrying the human gene for the poliovirus receptor (15, 20, 34). Thus, the IRES seems to be an important regulatory element for strain-specific expression of poliovirus neurovirulence. These two animal models show no difference in the development of the disease, even though replication of the virus in vivo must involve a number of biological interactions between viral and host factors. These results suggest that host factors of monkeys and mice, including IRES-related factors, support the expression of poliovirus neurovirulence (replication) in much the same way. However, it is possible that species differences between the IRES-related host factors of monkeys and mice exist.Several mutants with alterations in the stem-loop II (SLII) region were constructed from an infectious cDNA clone of the virulent Mahoney strain of poliovirus type 1 (39). The mutants replicated well in primate cells and in the CNS of monkeys but did poorly in mouse cells expressing human poliovirus receptor and in the CNS of the Tg mice carrying the human PVR gene (39). The replication of the mutant strains in mouse cells was blocked at the IRES-dependent translation initiation step, indicating that the function of the SLII as a part of the IRES is deficient in mouse cells but still active in primate cells. These differences in how the SLII mutants acted in the two animal models point to an interaction between the SLII and SLII-related host factors that could be a determinant for host-specific replication of poliovirus.To gain a deeper insight into the molecular basis of the function of the SLII region within the IRES, revertants that acquired the ability to replicate in mouse cells were isolated from the SLII mutants. Genetic analysis of mutation sites in the revertant genomes revealed that nt 107 within the 5′ noncoding region of poliovirus RNA influenced the efficiency of the IRES-dependent translation initiation process and that the remaining mutation sites (nt 120 to 161), in addition to nt 107, were required for the expression of the full revertant phenotype.     

Only One 3′-Hydroxyl Group Of ppp 5′A 2′p 5′A 2′p 5′A(2–5A) is Required for Activation of the Mouse 2–5A-Dependent Endonuclease

Abstract

The 3′-hydroxyl groups of each of the adenosines of 2–5A triraer (ppp5′A2′p5′A2′p5′A) were sequentially replaced by hydrogen through a phosphotriester synthetic approach. Biochemical evaluation of these analogs led to the conclusion that only the 3′-hydroxy group of the second adenosine is required for activation of RNase L.     

The preparation of a photoreactive analogue of 2′,3′-dideoxyuridine 5′-triphosphate and its use for photoaffinity modification of human replication protein A

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2′,3′-dideoxyuridine 5′-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2′-deoxyuridine 5′-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase β and to terminate DNA synthesis. A 5′-³²P-labeled primer with a photoreactive group at the 3′-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.     

A New 5′-Protecting Group for Use in Solid-Phase Synthesis of Oligoribonucleotides

Abstract

The 2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl (DNBSB) group is proposed as a protecting group for the 5′-position of nucleosides. The DNBSB group may be removed under mild non-acidic conditions and may have potential in solid-phase synthesis of oligoribo- and oligodeoxyribonucleotides.     

A Biotin Phosphoramidite Reagent for the Automated Synthesis of 5′-Biotinylated Oligonucleotides

Abstract

A bis(DMTr)biotin phosphoramidite containing serine and 6-aminohexanol moieties was prepared by a multiple-step reaction, and used successfully in the solid phase synthesis of 5′-biotinylated oligonucleotides.