Raj K. Bhatnagar

RNA Interference: Biology, Mechanism, and Applications

SUMMARY Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum

Summary Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double‐stranded RNA of cysteine protease genes (falcipain‐1 and ‐2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double‐stranded (ds)RNAs are reminiscent of the effects observed upon administering E‐64, a cysteine protease inhibitor. The parasites treated with falcipains dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs ≈ 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence‐specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

ABSTRACT RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.

Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac.

Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis.

Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis

ABSTRACT A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.

Normal exercise capacity in patients with severe left ventricular dysfunction: compensatory mechanisms.

About one-third of patients who have left ventricular dysfunction achieve normal levels of exercise. To elucidate the mechanisms that permit this to occur, we studied six patients with severe left ventricular dysfunction (average left ventricular ejection fraction 17 ± 2.5% [mean ± SEM]) who achieved nearly normal levels of exercise tolerance (greater than 11 mhutes of treadmill exercise, Sheffield protocol). All patients had normal pulmonary function at rest and during exercise. Hemodynamics were measured at rest and during supine and upright exercise. The major mechanisms of the preserved exercise capacity in these patients were chronotropic competence, ability to tolerate elevated wedge pressures (33 + 3 mm Hg) without dyspnea, ventricular dilation, and increased levels of plasma norepinephrine at rest and during exercise. Also, whereas peripheral vascular resistance was unchanged during supine exercise, it decreased by 50% during similar levels of upright exercise. As a consequence, increases in cardiac output from rest to exercise were greater during upright than supine exercise (100% vs 50%, respectively) (p < 0.05), and pulmonary wedge pressures were lower during upright than supine exercise (21 ± 5 mm Hg vs 33 + 3 mm Hg). Thus, multiple mechanisms permit some patients with severe left ventricular dysfunction to achieve normal levels of exercise. These studies emphasize that left ventricular function must be assessed by direct means rather than inferring function of the left ventricle from the results of an exercise tolerance test.

CORRELATION BETWEEN TYROSINE HYDROXYLASE ACTIVITY AND CATECHOLAMINE CONCENTRATION OR TURNOVER IN BRAIN REGIONS

Tyrosine hydroxylase activity correlated significantly with norepinephrine concentration and turnover, when results from regions containing predominantly noradrenergic terminals were compared, and with dopamine concentration and turnover when results from regions containing predominantly dopaminergic terminals were compared. Regions containing dopamine or norepinephrine cell bodies were characterized by higher tyrosine hydroxylase activities as compared to regions containing mostly nerve terminals. Higher levels of tyrosine hydroxylase activity and transmitter turnover were observed in regions containing dopaminergic terminals than in regions containing norepinephrine terminals. These findings are consistent with the view that tyrosine hydroxylase activity is linked to rates of catecholamine utilization by neurons in the CNS.

Chronic naltrexone increases opiate binding in brain and produces supersensitivity to morphine in the locus coeruleus of the rat

Rats were implanted subcutaneously for 2-4 weeks with slow-release pellets of naltrexone (10 mg) or placebo and then the pellets were removed. One day after removal of the pellet, animals were either (1) sacrificed and various CNS regions examined for specific binding of [3H]naloxone, [3H]etorphine or [3H]rauwolscine or (2) they were anesthetized and prepared acutely for assessing morphine-induced changes in the spontaneous activity of neurons in the locus coeruleus (LC). Naltrexone treatment significantly increased the number of specific binding sites for opiates, but not for alpha 2-adrenergic antagonists, in spinal cord, hypothalamus, striatum and cortex. Specific binding of [3H]naloxone was also increased in the LC. The spontaneous activity of neurons in the LC was reduced by the chronic naltrexone treatment, suggesting that these neurons became supersensitive to the tonic inhibitory effect of endogenous opioid peptides. Moreover, neurons in the LC of chronic naltrexone-treated rats exhibited an enhanced response to the inhibitory effects of morphine administered systemically. These results demonstrate that chronic opiate receptor blockade increases the number of receptor sites for morphine and that this increase in receptors is accompanied by a neuronal supersensitivity in the LC to morphine which can be assessed electrophysiologically.

Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

ABSTRACT Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer

RNA silencing is a conserved pathway that functions as an antiviral mechanism. The majority of viruses encode silencing suppressors that interfere with siRNA‐and miRNA‐guided silencing pathways. The insect flock house virus B2 protein (FHVB2) functions as an RNAi silencing suppressor that inhibits siRNA biogenesis. Here, we describe the generation of a GFP silent sensor line (Sf21) and a GFP sensor line expressing FHVB2 to study RNAi suppression mechanisms. Overexpression of FHVB2 resulted in suppression of GFP‐RNAi and resumption of GFP expression. Protein fractionation studies with FHVB2‐transfected cells showed that FHVB2 associates with ahigh‐molecular‐weight complex of Dicer and dsRNA/siRNAs. Yeast two‐hybrid and pulldown assays revealed an interaction between FHVB2 and Drosophila Dicer proteins that appeared to involve PAZ domains. To map the FHVB2 domains interacting with Dicer, we used a 17‐residue C‐terminal deletion mutant. RNAi suppression was reversed in cells transfected with the FHVB2 mutant as revealed by loss of GFP. Additional yeast two‐hybrid and in vitro pulldown assays confirmed that the C‐terminal region of FHVB2 was involved in the interaction with the PAZ domains of Dicers. These results thus reveal a novel interaction between FHVB2 and Dicer that leads to suppression of siRNA biogene‐sis.—Singh, G.,Popli, S., Hari, Y., Malhotra, P., Mukherjee, S., Bhatnagar, R.K. Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer. FASEB J. 23, 1845–1857 (2009)