Endogenous RNA Interference Research Articles

Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica

Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a “trigger” to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the serum and tissue expression of miR 21 and miR 199-a to be applied as early detectors for HCC. MethodsA total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-21 and miR-199-a) were detected by real time quantitative reverse-transcriptase PCR (RT-qPCR) in sera and tissues of chronic hepatitis and HCC patients. Besides, miR-21 and miR-199-a levels in relation to clinical and pathological factors were explored. ResultsWe found that the expression of serum miR-21 was distinctly increased in HCC compared with chronic hepatitis (P<0.001). miR 199-a was distinctly decreased in HCC compared with chronic hepatitis (P<0.001). In addition, median of miR 21 was increased in malignant when compared to adjacent non-malignant tissues without significant differences (P=0.191) while miR 199-a was significantly decreased in malignant when compared to adjacent nonmalignant tissues (P<0.001). ROC analysis showed that miR-21 and miR-199-a might be potential biomarkers for HCC. ConclusionsIn conclusion, the expression of miR-21 was significantly up-regulated and miR-199-a was significantly down regulated in serum of patients with HCC. Due to their reasonable sensitivity and specificity for disease progression, miR-21 and miR-199-a could be used as potential circulating biomarkers for HCC.

Application of Carbon Nanomaterials in Gene Delivery for Endogenous RNA Interference In Vitro and In Vivo.

Knocking down expression by small interfering RNA (siRNA) has shown high affinity, specificity and potency in silencing target gene sites. For effective endogenous RNA interference (RNAi), proper siRNA delivery vehicles are essential, to take the siRNA inside cells and protect them during the circulation. Carbon nanomaterials (CNMs) have been successfully applied in biomedicine and biosensor based on their ultra-high surface functionalization and nucleic acid molecular loading capacity. Recently, CNMs have drawn considerable research interest and expectation as potential non-viral vectors for siRNA delivery. Here we reviewed the recent application of CNMs in gene delivery for RNAi, mainly about fullerenes, carbon nanotubes (CNTs) and graphene.

MicroRNA-7 directly targets insulin-like growth factor 1 receptor to inhibit cellular growth and glucose metabolism in gliomas.

BackgroundRecent studies observed that altered energy metabolism has become widespread in cancer cells along with other cancer-associated traits that have been accepted as hallmarks of cancer. Akt signaling pathway is involved in the aerobic glycolysis program. However, mechanisms underlying the regulation of aerobic glycolysis and Akt activity in gliomas remain unclear. MicroRNAs are a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of targeted genes. This study was conducted to detect the function of miR-7 targeting insulin-like growth factor 1 receptor (IGF-1R), which is an upstream regulator of Akt.MethodsMicroRNA expression data for gliomas and normal controls were downloaded from The Cancer Genome Atlas (TCGA) database. Quantitative real-time PCR was used to measure the microRNA-7 (miR-7) expression level, and Western blot was performed to detect protein expression in U87 and U251 cells. Colony formation assay and glycolysis stress test were also conducted. Luciferase reporter assay was used to identify the mechanism of IGF-1R and miR-7 regulation.ResultsmiR-7 was downregulated in human glioma tissues based on TCGA database. Forced expression of miR-7 or IGF-1R knockdown inhibited colony formation and glucose metabolic capabilities of glioma cells in vitro and decreased the p-Akt expression level. Bioinformatics analysis results indicated that IGF-1R could be a target of miR-7. Western blot and luciferase reporter assays showed that miR-7 modulated IGF-1R expression by directly targeting the binding site within the 3′-untranslated region.ConclusionsThis study provides the first evidence that miR-7 inhibits cellular growth and glucose metabolism in gliomas, at least partially, by regulating the IGF-1R/Akt signaling pathway. Therefore, miR-7 is a promising molecular drug for glioma treatment.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_211

Serum microRNA 143 and microRNA 215 as potential biomarkers for the diagnosis of chronic hepatitis and hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis or monitor postoperative recurrence and to give adequate treatment for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the application of miR-143 and miR-215 in the diagnosis of HCC.MethodsA total of 340 serum samples (127 samples from controls, 118 samples from hepatitis and 95 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-143 and miR-215) were detected by probe-based stem-loop quantitative reverse-transcriptase PCR (RT-qPCR) in controls, hepatitis and HCC patients. Besides, the relationship between miR-143 and miR-215 levels and clinical and pathological factors was explored.ResultsWe found that the expression of serum miR-215 was distinctly increased in chronic hepatitis compared with controls (mean ± SD: 6.79 ± 0.72 vs. 3.46 ± 0.37, P < 0.001 and mean ± SD: 8.38 ± 0.87 vs. 3.46 ± 0.37, P < 0.001). In addition, we conduct ROC analyses to detect the potential application of miR-143 and miR-215 in the diagnosis of chronic hepatitis and HCC. Our results showed that miR-143 and miR-215 might be a potential biomarker for the hepatitis and HCC.ConclusionsIn conclusion, the expression of miR-143 and miR-215 in serum were significantly up-regulated in patients with chronic hepatitis and HCC. Due to its reasonable sensitivity and specificity for both diseases, miR-143 and miR-215 could be as potential circulating biomarkers.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1048932281272754

Cationic nanostructured polymers for siRNA delivery in murine calvarial pre-osteoblasts.

The endogenous RNA interference (RNAi) pathway enables control of pathologies caused by the dysregulation of proteins. Several biological molecules are active in RNAi including short interfering ribonucleic acid (siRNA). The effective utilization of siRNA as a therapeutic agent has been marked with distinct challenges, namely in intracellular delivery and achieving a sufficient dosage to affect protein expression. A delivery strategy we have developed to improve safety and efficacy of siRNA includes complexing siRNA with nanostructured polymers delivery systems (NSPs). These NSPs are synthesized via atom transfer radical polymerization (ATRP) and combine several important advances in polymer architecture for siRNA delivery. This includes shielding the cationic charge of the NSP with a poly(ethylene glycol) (PEG) shell to promote cell viability in MC3T3-E1.4 pre-osteoblasts, and minimize the inflammatory response in a C57BL/6 mouse model. In our gene knockdown experiments targeting glyceraldehyde 3-phosphate dehydrogenase Gapdh expression, star polymer and nanogel polyplexes suppressed Gapdh mRNA to levels comparable to cells treated with Lipofectamine RNAiMAX lipoplexes.

Renaissance of mammalian endogenous RNAi.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by long double-stranded RNA (dsRNA). RNAi is an ancient eukaryotic defense mechanism against viruses and mobile elements. In mammals, endogenous RNAi was outstripped during evolution by the current innate and acquired immunity. The RNAi apparatus, which remains essentially intact, serves mostly the microRNA pathway, which regulates endogenous gene expression. Remarkably, several recent publications brought the mammalian endogenous RNAi pathway back into the spotlight. Here, I will provide an up-to-date review of the mammalian endogenous RNAi pathway with a focus on its defensive role and overlaps with miRNA and piRNA pathways.

Endogenous RNA interference is driven by copy number

A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001.

Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform

RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, β-cell differentiation, normal β-cell function and pancreatic cancer. Our aim is to review the process of identifying PDX1 as a specific, potential RNAi target in pancreatic cancer, as well as the underlying mechanisms and various forms of RNAi, with subsequent testing and development of PDX1-targeted bifunctional shRNA therapy.

SYL040012, a siRNA for the treatment of glaucoma

AbstractPurpose SYL040012 is a siRNA under developed for the treatment of glaucoma and increased intraocular pressure (IOP). siRNAs are small double stranded RNAs that regulate protein expression at transcription level using the endogenous RNA interference machinery. Therapeutically siRNAs are attractive due to their specificity, potency and ability to silence targets that are not addressable by small molecules. SYL040012 targets ADRB2, a well‐known target for glaucoma.Methods Silencing activity of SYL040012 was validated in cell lines and IOP lowering efficacy was studied in rabbit. Silencing activity was assessed by extracting total RNA from cells or animal tissues and analyzed by real time‐PCR. IOP was measured using applanation tonometry. Tissue and plasma exposure was evaluated following a single instillation to rabbits using a non‐denaturing Anion Exchange HPLC combined with fluorescence detection. 28‐day regulatory toxicology studies were performed in cynomolgus monkeys following a daily ocular instillation of one of three doses of SYL040012 or vehicle. Phase I clinical trials were performed in healthy volunteers or individuals with increased IOP that received either a single dose or seven repeated administrations of SYL040012.Results SYL040012 reaches structures of the eye relevant to the treatment of glaucoma when administered in eye drops; but is only detected in systemic circulation and non‐ocular tissues at trace levels. Presence of this compound in the ciliary body it results in specific down‐regulation of the target gene and reduction in IOP in animal models. Preclinical and clinical studies show that SYL040012 is well tolerated both systemically and locally.Conclusion In summary, SYL040012 is a promising candidate for the treatment of increased intraocular pressure associated to glaucoma. Commercial interest

Abstract B11: Coupling between the DNA methylation and the alternative splicing

Abstract Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally and chromatin-borne information participates in alternative splicing. We have found recently that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) and the chromodomain protein HP1γ are a characteristic of the alternative exons of several genes including CD44 (Saint-André et al. 2011). On this gene, we have shown that the endogenous RNA interference pathway is involved in the H3K9me3 targeting on the variant exons through the recruitment of the nuclear argonaute proteins (AGO1 or AGO2) (Ameyar-Zazoua et al. 2012). Because the DNA methylation is an epigenetic mark accompanying the transcriptional silencing, we now explore whether the DNA modification into the active genes could play a role in the regulation of the alternative splicing. Studying the transcriptome and the genome of murine ES cells, allowed us to show that the enzymes that methylate DNA (DNMT1, 3a and 3b) regulate the splicing of many genes. In Hela cells the DNMT1 depletion causes a decrease in the inclusion of alternative exons of CD44. Similar results were obtained after depletion of MBD1 and MBD2, two proteins that bind to methylated DNA. Together these results suggest that DNA methylation regulates splicing of CD44 by a direct mechanism involving MBD1 and MBD2. The disregulation of the DNA methylation on CpGs is a hallmark of the tumorigenesis. However, the effect of DNA methylation changes within coding regions of the genes is poorly understood. We used the well-known model of breast tumor progression based on the MCF10A cells, to investigate the changes into the DNA methylation of the CD44 coding region. We found that the intragenic DNA methylation is modified between the MCF10A parental cells and the derived DCIS and CA1A transformed cells and this is correlated with the inclusion of the CD44 alternative exons. Altogether these results suggest that modifications of DNA methylation could contribute to the disregulations of alternative splicing decisions contributing to the tumor progression. Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau JC, Mathieu J, Hamiche A, Ait-Si-Ali S, Muchardt C, Harel-Bellan A, Batsché E (2012) Argonaute proteins couple chromatin silencing to alternative splicing. Nat. Struc. Mol. Biol 19:998-1004. Saint-André V., Batsché E., Rachez C. and Muchardt C. (2011) Histone H3 lysine 9 tri-methylation and HP1γ favor inclusion of alternative exons. Nat. Struc. Mol. Biol 18:337-44. Citation Format: Oriane Mauger, Christian Muchardt, Eric Batsché. Coupling between the DNA methylation and the alternative splicing. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B11.

Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90 % identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9716-5) contains supplementary material, which is available to authorized users.

Abstract 3205: In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system.

Abstract Background: Small RNAs are able to suppress expression of any gene through the endogenous RNA interference pathway and have been used to identify therapeutic targets in a variety of pathological conditions. In this project, we aimed to optimise a lentiviral inducible-knockdown small hairpin RNA (shRNA) system for efficient in vivo cancer target validation, using polo-like kinase 1 (PLK1), an essential cell-cycle protein, as a proof-of-concept target. Methods: An inducible shRNA construct targeting PLK1 or a control construct was transduced into the colon carcinoma cell line SW620. Inducible shRNA expression (marked by turbo-RFP) was monitored by fluorescence microscopy and flow cytometry in vitro and by spectrum CT in vivo. PLK1 gene expression was quantified by real-time PCR and protein expression was quantified by Western blotting in vitro or IHC ex vivo. Cell viability was assessed by MTT assays. Xenografts were established by subcutaneous injection of PLK1 inducible-knockdown cells into nude mice. Growth inhibitory effect was assessed by calliper measurement of tumour size and correlated with ex vivo assessment of gene and protein knockdown, and of cell proliferation as measured by Ki67 staining. Results: Cell viability assays demonstrated that concentrations of 2ug/ml of doxycycline or less did not result in cell death over an assay period of 7 days in both wild-type cells and cells transduced with a control shRNA construct. Following doxycycline treatment, PLK1 inducible-knockdown cells showed dose- and time-dependent shRNA induction with an increase in the level of shRNA expression and the percentage of positive cells. PLK1 down-regulation correlated with induction of shRNA expression reaching 80% gene knock-down and 60% protein knock-down 72 hours post-induction. The optimal delivery route of doxycycline via measurement of turbo-RFP induction and PK/PD measurements was assessed in vivo. In tumour-bearing mice treated with doxycycline, there was a decline in tumour growth rate in the PLK1 knockdown group compared with the control group. Ex vivo analysis showed significantly lower PLK1 gene and protein expression in the doxycycline-treated group compared with the control group which correlated with lower rates of cell proliferation in the treated group. Conclusion: The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient target validation methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system. Citation Format: Yinfei Yin, Rajendra Kumari, Siddharth Subramaniam, Peter King, Sue Watson, Philip Mallinder, Anna Grabowska. In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2013-3205

Advances in RNA Interference Technology and Its Impact on Nutritional Improvement, Disease and Insect Control in Plants

This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

BackgroundAt the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs).ResultsTo test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs.ConclusionsOur data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself.

The Therapeutic Potential of RNA Interference: Novel Approaches for Cancer Treatment

Exo- and endogenous RNA interference (RNAi) is a recently developed post-transcriptional gene silencing system and is regarded as one of the most effective and specific gene silencing techniques with therapeutic potential. siRNA technology is also expected to be an invaluable treatment tool for viral infections, dominant disorders, neurological disorders, and cancers. Novel and increasingly effective therapies are urgently needed as conventional radio/chemotherapy is of limited efficacy in advanced stages of some malignant diseases. In some cancers, it is known that resistance to chemotherapy is mainly attributed to increased expression of anti-apoptotic genes, e.g. members of the bcl-2 family that stabilize the mitochondrial membrane. The siRNA-mediated knockdown of bcl-2 leads to pronounced anti-tumor effects in a pancreatic cancer model, especially in combination with chemotherapy even at otherwise ineffective concentrations. The future success of this approach will depend on the development of effective, specific, and safe delivery systems. In addition to therapeutic RNAi, endogenous RNAi processes may also contribute to cancer development. For example, miRNAs have been shown to suppress target gene expression through binding to the 3'-untranslated regions (3'-UTR) of target mRNAs. miRNAs control many functions in regard to cell viability, including differentiation, proliferation, and apoptosis, especially during cancer progression. More investigation into miRNAs will lead to new diagnostic and therapeutic approaches in the near future.

Gene targeting in primary human trophoblasts

Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts.

Differential Impact of the HEN1 Homolog HENN-1 on 21U and 26G RNAs in the Germline of Caenorhabditis elegans

RNA interference (RNAi)–related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3′ ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA–methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3′-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1–bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4–bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

BackgroundIntegrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions.Principal FindingsTo establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained.ConclusionIn summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.

Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi

Endogenous RNA interference (endo-RNAi) pathways use a variety of mechanisms to generate siRNA and to mediate gene silencing. In Caenorhabditis elegans, DCR-1 is essential for competing RNAi pathways-the ERI endo-RNAi pathway and the exogenous RNAi pathway-to function. Here, we demonstrate that DCR-1 forms exclusive complexes in each pathway and further define the ERI-DCR-1 complex. We show that the tandem tudor protein ERI-5 potentiates ERI endo-RNAi by tethering an RNA-dependent RNA polymerase (RdRP) module to DCR-1. In the absence of ERI-5, the RdRP module is uncoupled from DCR-1. Notably, EKL-1, an ERI-5 paralog that specifies distinct RdRP modules in Dicer-independent endo-RNAi pathways, partially compensates for the loss of ERI-5 without interacting with DCR-1. Our results implicate tudor proteins in the recruitment of RdRP complexes to specific steps within DCR-1-dependent and DCR-1-independent endo-RNAi pathways.