Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Gautam Buddha University

Retroviruses rely on host RNA-binding proteins to modulate various steps in their replication. Previously several animal retroviruses were determined to mediate Dhx9/RNA helicase A (RHA) interaction with a 5' terminal post-transcriptional control element (PCE) for efficient translation. Herein PCE reporter assays determined HTLV-1 and HIV-1 RU5 confer orientation-dependent PCE activity. The effect of Dhx9/RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1(NL4-3) provirus determined that RHA is necessary for efficient HIV-1 RNA translation and requires ATPase-dependent helicase function. Quantitative analysis determined HIV-1 RNA steady-state and cytoplasmic accumulation were not reduced; rather the translational activity of viral RNA was reduced. Western blotting determined that RHA-deficient virions assemble with Lys-tRNA synthetase, exhibit processed reverse transcriptase and contain similar level of viral RNA, but they are poorly infectious on primary lymphocytes and HeLa cells. The results demonstrate RHA is an important host factor within the virus-producer cell and within the viral particle. The identification of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism.

RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions.

RNase P, which catalyzes tRNA 5'-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR's cis cleavage of precursor tRNA(Gln) (pre-tRNA(Gln)), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR's mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR's apparent rate of correct cleavage by 11,140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR's rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5'-maturation of pre-tRNA(Gln) and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5'-processing.

Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors.

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5-7 days in the disease development as compared to wild-type plants.

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene.

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

Oxidative stress is implicated in toxicant-induced inflammation leading to chronic diseases. Polymorphonuclear leukocytes (PMNs) offer the first line of defense against infection in the mammals and protect against inflammation-mediated pathological anomalies. Conversely, activated PMNs contribute to the oxidative stress-mediated damage and inflammation. The study aimed to investigate the status of oxidative stress and antioxidant defense system in the PMNs of rats treated with/without zinc (Zn) and/or paraquat (PQ) in the presence or absence of a synthetic superoxide dismutase/catalase mimetic, a manganese-salen compound-EUK-134 and/or a glutathione precursor, N-acetyl cysteine (NAC). While Zn and/or PQ elevated the total free radical generation, lipid peroxidation (LPO) and catalytic activity of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase alpha 4-4 (GSTA4-4), a pronounced decrease in reduced glutathione (GSH) and glutathione reductase (GR) activity was also observed. Zn and/or PQ augmented the expression of metallothionein-I and II and GSTA4-4. Pre-treatment of EUK-134 or NAC alone altered the level of total free radical generation, LPO, GSH content and catalytic activity of MPO, SOD, GR and GPx and the expression of metallothionein I and II towards normalcy. The alterations were more pronounced in the PMNs of rats treated with EUK-134 and NAC in combination. Catalytic activity/expression of GSTA4-4 remained unchanged in the PMNs of EUK-134 or NAC treated rats. The results demonstrate that EUK-134 and NAC protect PMNs from the toxic effects of Zn and PQ in rats and also suggest that metallothioneins I/II might contribute to antioxidant defense under GSH depleted conditions.

The manganese-salen compound EUK-134 and N-acetyl cysteine rescue from zinc- and paraquat-induced toxicity in rat polymorphonuclear leukocytes.

Accumulation of zinc (Zn) in dopaminergic neurons is implicated in Parkinson's disease (PD), and microglial activation plays a critical role in toxin-induced Parkinsonism. Oxidative stress is accused in Zn-induced dopaminergic neurodegeneration; however, its connection with microglial activation is still not known. This study was undertaken to elucidate the role and underlying mechanism of microglial activation in Zn-induced nigrostriatal dopaminergic neurodegeneration. Male Wistar rats were treated intraperitoneally with/without zinc sulphate (20 mg/kg) in the presence/absence of minocycline (30 mg/kg), a microglial activation inhibitor, for 2-12 weeks. While neurobehavioral and biochemical indexes of PD and number of dopaminergic neurons were reduced, the number of microglial cells was increased in the substantia nigra of the Zn-exposed animals. Similarly, Zn elevated lipid peroxidation (LPO) and activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; however, catalase activity was reduced. Besides, Zn increased an association of NADPH oxidase subunit p67(phox) with membrane, cytochrome c release from the mitochondria and cleavage of pro-caspase 3. Zn attenuated the expression of tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT-2) while augmented the expression of dopamine transporter (DAT) and heme oxygenase-1 (HO-1). Minocycline alleviated Zn-induced behavioural impairments, loss of TH-positive neurons, activated microglial cells and biochemical indexes and modulated the expression of studied genes/proteins towards normalcy. The results demonstrate that minocycline reduces the number of activated microglial cells and oxidative stress, which rescue from Zn-induced changes in the expression of monoamine transporter and nigrostriatal dopaminergic neurodegeneration.

Minocycline Rescues from Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration: Biochemical and Molecular Interventions.

Oxidative stress is a key factor in Parkinson's disease (PD) pathogenesis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and impaired mitochondrion regulate oxidative stress-mediated maneb (MB)- and paraquat (PQ)-induced Parkinsonism. However, their contribution in the MB- and PQ-induced toxicity in polymorphonuclear leukocytes (PMNs) is still elusive. The study investigated the role of NADPH oxidase and mitochondria in MB- and/or PQ-induced oxidative stress in the PMNs and the crossing point between the two. Animals were treated with MB and/or PQ for 1-3 weeks along with respective controls. In a few sets of experiments, rats were treated with/without NADPH oxidase inhibitor, apocynin, an hour prior to MB and/or PQ treatment. PMNs of MB and/or PQ treated animals were also treated with/without carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to assess the role of the mitochondria in superoxide and total free radical productions. MB and/or PQ were found to increase the level of total reactive oxygen species (ROS), superoxide radicals, catalytic activity and expression of NADPH oxidase and superoxide dismutase (SOD1/2) and mitochondrial ROS content in a time dependent manner. Conversely, catalase activity and mitochondrial membrane potential were attenuated. Apocynin alleviated MB- and/or PQ-induced changes in total ROS, superoxide radicals, expression/catalytic activity of NADPH oxidase and SOD1/2 along with the mitochondrial ROS and membrane potential. CCCP also inhibited ROS and superoxide levels in the PMNs of MB and/or PQ-treated animals. The results demonstrate the involvement of NADPH oxidase and mitochondrial dysfunction in MB and PQ-induced oxidative stress in PMNs and a plausible crosstalk between them.

NADPH oxidase mediated maneb- and paraquat-induced oxidative stress in rat polymorphs: Crosstalk with mitochondrial dysfunction.

Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.

HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

Genome sequencing has not only extended our understanding of the blueprints of many plant species but has also revealed the secrets of coding and non-coding genes. We present here a brief introduction to and personal account of key RNA-based technologies, as well as their development and applications for functional genomics of plant coding and non-coding genes, with a focus on short tandem target mimics (STTMs), artificial microRNAs (amiRNAs), and CRISPR/Cas9. In addition, their use in multiplex technologies for the functional dissection of gene networks is discussed.

Essential RNA-Based Technologies and Their Applications in Plant Functional Genomics.

The study aimed to investigate the role of NO and neuronal NO synthase (nNOS) in Zn-induced neurodegeneration. Animals were treated with zinc sulfate (20 mg/kg), twice a week, for 2-12 weeks along with control. In a few sets, animals were also treated with/without a NO donor, sodium nitroprusside (SNP), or S-nitroso-N-acetyl penicillamine (SNAP) for 12 weeks. Moreover, human neuroblastoma (SH-SY-5Y) cells were also employed to investigate the role of nNOS in Zn-induced toxicity in in vitro in the presence/absence of nNOS inhibitor, 7-nitroindazole (7-NI). Zn caused time-dependent reduction in nitrite content and total/nNOS activity/expression. SNP/SNAP discernibly alleviated Zn-induced neurobehavioral impairments, dopaminergic neurodegeneration, tyrosine hydroxylase (TH) expression, and striatal dopamine depletion. NO donors also salvage from Zn-induced increase in lipid peroxidation (LPO), mitochondrial cytochrome c release, and caspase-3 activation. While Zn elevated LPO content, it attenuated nitrite content, nNOS activity, and glutathione level along with the expression of TH and nNOS in SH-SY-5Y cells. 7-NI further augmented Zn-induced changes in the cell viability, oxidative stress, and expression of TH and nNOS. The results obtained thus demonstrate that Zn inhibits nNOS that partially contributes to an increase in oxidative stress, which subsequently leads to the nigrostriatal dopaminergic neurodegeneration.

Neuronal Nitric Oxide Synthase Negatively Regulates Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration.

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

Deepali Singh

School of Biotechnology

Gautam Buddha University

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Deepali Singh

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Biotechnology

Gautam Buddha University

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

School of Biotechnology