Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis

Although inhibition of RNA interference (RNAi) by plant virus proteins has been shown to enhance viral replication and pathogenesis in plants, no viral gene product has as yet been shown to inhibit RNAi in vertebrate cells. Here, we present evidence demonstrating that the highly structured approximately 160-nucleotide adenoviral VA1 noncoding RNA can inhibit RNAi at physiological levels of expression. VA1, which is expressed at very high levels in adenovirus-infected cells, potently inhibited RNAi induced by short hairpin RNAs (shRNAs) or human microRNA precursors but did not affect RNAi induced by artificial short interfering RNA duplexes. Inhibition appeared to be due both to inhibition of nuclear export of shRNA or premicro-RNA precursors, competition for the Exportin 5 nuclear export factor, and inhibition of Dicer function by direct binding of Dicer. Together, these data argue that adenovirus infection can result in inhibition of RNAi and identify VA1 RNA as the first viral gene product able to inhibit RNAi in human cells.     

TAK1siRNA对C2C12细胞内TAK1基因表达的影响

目的:高效下调TAK1基因表达的小干扰RNA(siRNA)分子的获得.方法:采用脂质体转染方法,将3对(siRNA ID#94455、siRNA ID # 94549、siRNA ID # 189006)人工合成的TAK1基因特异的小干扰RNA(siRNA)分子分别导入小鼠成肌细胞C2C12中,采用实时荧光定量PCR方法分析细胞内TAK1基因的相对表达.结果:和对照组相比,siRNA ID # 94455、siRNA ID # 94549和siRNA ID # 189006分别下调了细胞内TAK1基因的mRNA表达水平33.34%、46.73%和79.97%.结论:实验获得了能够高效下调TAK1基因表达的siRNA.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.     

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.     

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence, dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3‘ overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNAduplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0-450 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5‘ end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.     

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.     

Short interfering RNA-directed inhibition of hepatitis B virus replication

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of mRNA. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. Human hepatoma cells were transfected with HBV DNA and siRNA against HBV-pregenome RNA. Transfection experiments demonstrated that the siRNA reduced the amount of HBV-pregenome RNA and resulted in reduction of the levels of replicative intermediates and viral protein. Our results indicate that siRNA-mediated gene silencing inhibits HBV replication through suppression of viral RNA, which may be useful as a potential therapeutic modality.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.     

Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element

RNA interference (RNAi) is a powerful method for suppressing the expression of a gene of interest, and can be induced by 21-25 nucleotide small interfering RNA (siRNA) duplexes homologous to the silenced gene, which function as sequence-specific RNAi mediators in RNA-induced silencing complexes (RISCs). In the previous study, it was shown that fork-siRNA duplexes, whose sense-stranded siRNA elements carried a few nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, could enhance RNAi activity more than conventional siRNA duplexes in cultured mammalian cells. In this study, we further characterized fork-siRNA duplexes using reporter plasmids carrying target sequences complementary to the sense- or antisense-stranded siRNA elements in the untranslated region of Renilla luciferase. The data presented here suggest that nucleotide mismatches at either the 3'- or 5'-end of the sense-stranded siRNA elements in fork-siRNA duplexes could influence assembly of not only the antisense-stranded siRNA elements but also the sense-stranded elements into RISCs. In addition, we further suggest the possibility that there could be a positional effect of siRNA duplex on RNAi activity.     

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.     

STUB1 regulates antiviral RNAi through inducing ubiquitination and degradation of Dicer and AGO2 in mammals

RNA interference (RNAi) is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms. However, the mechanism by which antiviral RNAi in mammals is regulated is poorly understood. In this study, we uncovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1) was a new regulator of the RNAi machinery in mammals. We found that STUB1 interacted with and ubiquitinated AGO2, and targeted it for degradation in a chaperon-dependent manner. STUB1 promoted the formation of Lys48 (K48)-linked polyubiquitin chains on AGO2, and facilitated AGO2 degradation through ubiquitin-proteasome system. In addition to AGO2, STUB1 also induced the protein degradation of AGO1, AGO3 and AGO4. Further investigation revealed that STUB1 also regulated Dicer's ubiquitination via K48-linked polyubiquitin and induced the degradation of Dicer as well as its specialized form, termed antiviral Dicer (aviDicer) that expresses in mammalian stem cells. Moreover, we found that STUB1 deficiency up-regulated Dicer and AGO2, thereby enhancing the RNAi response and efficiently inhibiting viral replication in mammalian cells. Using the newborn mouse model of Enterovirus A71 (EV-A71), we confirmed that STUB1 deficiency enhanced the virus-derived siRNAs production and antiviral RNAi, which elicited a potent antiviral effect against EV-A71 infection in vivo. In summary, our findings uncovered that the E3 ubiquitin ligase STUB1 was a general regulator of the RNAi machinery by targeting Dicer, aviDicer and AGO1–4. Moreover, STUB1 regulated the RNAi response through mediating the abundance of Dicer and AGO2 during viral infection, thereby providing novel insights into the regulation of antiviral RNAi in mammals.     

Potent RNAi by short RNA triggers

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3′-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an ~42 Å A-form helix with ~1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.     

Lipopolysaccharide-induced monocyte chemotactic protein-1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.