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PubMed2006

How golden is silence? Teaching undergraduates the power and limits of RNA interference.

It is hard and getting harder to strike a satisfying balance in teaching. Time dedicated to student-generated models or ideas is often sacrificed in an effort to "get through the syllabus." I describe a series of RNA interference (RNAi) experiments for undergraduate students that simultaneously explores fundamental concepts in gene regulation, develops cutting-edge laboratory skills, and embraces student-directed learning. Students design a small interfering RNA (siRNA) against luciferase, add it to cells expressing this gene, and then quantitatively assess the siRNA's effect on both intended and unintended targets, using a luciferase assay and a DNA microarray. Because both RNAi and microarray technologies are relatively new, with no clear consensus on their analysis or limitations, students are encouraged to explore different approaches to the design of their reagents and interpretations of their data. The ability to creatively formulate a hypothesis-driven experimental approach to a scientific question and to critically evaluate collected data is stressed. Equally important, this experiment emphasizes how modern scientific ideas emerge, are debated, tested, and decided.

Educational Measurement↗
PubMed2006

Role of TIF1alpha as a modulator of embryonic transcription in the mouse zygote.

The first events of the development of any embryo are under maternal control until the zygotic genome becomes activated. In the mouse embryo, the major wave of transcription activation occurs at the 2-cell stage, but transcription starts already at the zygote (1-cell) stage. Very little is known about the molecules involved in this process. We show that the transcription intermediary factor 1 alpha (TIF1alpha) is involved in modulating gene expression during the first wave of transcription activation. At the onset of genome activation, TIF1alpha translocates from the cytoplasm into the pronuclei to sites of active transcription. These sites are enriched with the chromatin remodelers BRG-1 and SNF2H. When we ablate TIF1alpha through either RNA interference (RNAi) or microinjection of specific antibodies into zygotes, most of the embryos arrest their development at the 2-4-cell stage transition. The ablation of TIF1alpha leads to mislocalization of RNA polymerase II and the chromatin remodelers SNF2H and BRG-1. Using a chromatin immunoprecipitation cloning approach, we identify genes that are regulated by TIF1alpha in the zygote and find that transcription of these genes is misregulated upon TIF1alpha ablation. We further show that the expression of some of these genes is dependent on SNF2H and that RNAi for SNF2H compromises development, suggesting that TIF1alpha mediates activation of gene expression in the zygote via SNF2H. These studies indicate that TIF1alpha is a factor that modulates the expression of a set of genes during the first wave of genome activation in the mouse embryo.

Adenosine Triphosphatases↗
PubMed2003

siRNAs and shRNAs: skeleton keys to the human genome.

The use of RNA interference (RNAi) to evoke gene silencing in mammalian cell systems is rapidly becoming a standard laboratory technique. Recent experiments have also proven the utility of RNAi in animals. The next few years will bear witness to the first RNAi-based genome-wide screens in mammals, breaking the barrier to the use of mammalian cells as a genetic system. In this review, some of the key features of RNAi in mammalian systems are discussed.

Animals↗
PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗
PubMed2005

Amplification of RNAi--targeting HLA mRNAs.

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.

Base Sequence↗
PubMed2004

Genome-wide RNAi as a route to gene function in Drosophila.

With the sequencing of the human genome and the genomes of most major model organisms completed, the systematic characterisation of gene functions remains a key challenge. During the past few years, RNA interference (RNAi) has become a powerful tool to silence the expression of genes and analyse their loss-of-function phenotype when mutant alleles are not available. Genome-wide RNAi screens against all predicted genes have been successfully used to dissect a variety of biological processes in Caenorhabditis elegans. Recently, a genome-wide library of double-stranded RNAs, that target every gene in the Drosophila genome and that is suitable for high throughput cell-based assays, was published. In this paper, recent advances will be summarised. Screening strategies and applications as a route to comprehensively characterising gene function will be discussed.

Animals↗
PubMed2005

Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis.

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8-33% (Apaf-1) and to 30-50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17-33% (Apaf-1) and to 38-50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.

Apoptosis↗
PubMed2006

Element 1360 and RNAi components contribute to HP1-dependent silencing of a pericentric reporter.

In eukaryotes, distinct regions of the genome are packaged as euchromatin (less condensed, more active) or heterochromatin (condensed, silenced). Studies in yeast, plants, and flies suggest that RNA interference (RNAi) is linked to heterochromatin formation and transcriptional silencing of transposable element (TE) sequences. We previously reported that insertion of a mobile hsp70-white reporter within 10 kb of a 1360 element on chromosome four of Drosophila melanogaster correlates with variegation (silencing). Here, we report small RNAs (approximately 23 nt) corresponding to 1360, indicating processing by the RNAi machinery. To directly test the ability of 1360 to silence a nearby gene in vivo, we introduced a P element construct carrying a single copy of 1360 upstream of the hsp70-white reporter into flies. This 1360 element contributes to HP1-dependent variegation at a pericentric insertion site, as demonstrated by a decrease in silencing after FLP-mediated removal of 1360. In euchromatin, 1360 is not sufficient to induce silencing, suggesting that proximity to pericentric heterochromatin and/or a high local TE density contributes to heterochromatin formation. Silencing of the 1360, hsp70-white reporter is sensitive to mutations in RNAi components. Our results implicate 1360 as a target for sequence-specific heterochromatic silencing through an RNAi-dependent mechanism.

Animals↗
PubMed2006

RNA interference: big applause for silencing in Stockholm.

Eight years ago, Craig Mello, Andrew Fire, and their coworkers provided the first demonstration that double-stranded RNA (dsRNA) triggers the gene-silencing technique that we now call RNA interference (RNAi). For this landmark discovery, Mello and Fire are honored with this year's Nobel Prize in Physiology or Medicine.

Animals↗
PubMed2006

Coupling of transcription termination to RNAi.

In metazoans, the mechanisms of transcriptional termination by RNA polymerase II (Pol II) and accelerated decay of messenger RNA (mRNA) following transcription shutdown are linked by sharing the same sequence elements and mRNA elongation, processing and termination factors. This begs the question, how could one process have two opposite outcomes, making or degrading mRNA? An integrated "allosteric-GENEi-torpedo" model that could explain this paradox predicts participation of two novel factors: (1) An allosteric factor, regulated by a physiological repressor, binds to a unique sequence element of a gene near the site of cleavage and polyadenylation, poly(A) site, and acts on the homologous site on the nascent transcript to cause its cleavage. The conformational changes of this factor determine the fate of nascent RNA, either to get cleaved and processed to mature mRNA for directing protein synthesis, or not to get cleaved and become template for double-stranded (ds) RNA synthesis. (2) A general transcription termination factor, recruited by transcribing Pol II at the poly(A) site, allostrically alters and induces Pol II to switch template from DNA to nascent RNA several hundred nucleotides downstream of the poly(A) site. The template switch disengages Pol II from DNA and effectively terminates transcription. The Pol II with newly acquired RNA-dependent RNA polymerase activity retraces its path, back along the nascent RNA, so generating dsRNA. The extent to which it can retrace this path is determined by the factors influencing the cleavage of the pre-mRNA at the site of polyA addition. If cleavage and polyadenylation occur, the retracing is cut short, the 3' RNA is degraded by an exonuclease and the polymerase is liberated to reinitiate transcription. If the cleavage is inhibited, then a full-length dsRNA can be produced. This can then be subject to cleavage by "Dicer", which generates fragments of approximately 22bp that guide degradation of the cognate mRNA via the RNA interference (RNAi) pathway. This model complements the current "allosteric-torpedo" model of transcription termination, and could explain the apparent paradox of the divergent results of a common biological process.

DEAD-box RNA Helicases↗
PubMed2003

RNA interference for treating cancers caused by viral infection.

The newly discovered phenomenon of RNA interference (RNAi) offers the dual facility of selective viral gene silencing coupled with ease of tailoring to meet genetic variation within the viral genome. Such promise identifies RNAi as an exciting new approach to treat viral-induced diseases, including viral-induced cancers (e.g. cervical carcinoma, hepatocellular carcinomas and haematopoietic and lymphoid malignancies). Cervical cancer is the second most common cancer in women worldwide and is caused by human papilloma virus (HPV). Silencing of HPV gene expression by RNAi induces apoptosis of cervical carcinoma cells in culture, and the effect is apparent within 3 days. The RNAi process is triggered by double-stranded RNA (dsRNA), and a single dose is sufficient to maintain RNAi for several days in vitro (cell culture) and in vivo (experimental animal models). Thus, the stage is set for the development of novel antiviral and anticancer therapies based upon selective gene silencing by RNA interference.

Animals↗
PubMed2003

Expressing functional siRNAs in mammalian cells using convergent transcription.

BACKGROUND: The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing. RESULTS: With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. CONCLUSION: The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections.

Cell Line↗
PubMed2004

Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research.

Genes linked to human cancers often function in evolutionary conserved pathways, and research in C. elegans has been instrumental in dissecting some of the pathways affected, such as apoptosis and Ras signalling. The advent of RNA interference (RNAi) technology has allowed high-throughput loss-of-function analyses of C. elegans gene functions. Here we review some of the most recent genome-wide RNAi screens that have been conducted and discuss their impact on cancer research and possibilities for future screens. We also show that genes causally implicated in human cancers are significantly more likely to have a C. elegans homologue than average, validating the use of C. elegans as a cancer gene discovery platform. We foresee that genome-wide RNAi screens in C. elegans will continue to be productive in identifying new cancer gene candidates and will provide further insights into cancer gene functions.

Animals↗
PubMed2006

Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3'hExo/ERI-1 family of 3' to 5' exonucleases.

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.

Amino Acid Sequence↗
PubMed2003

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei.

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.

Animals↗
PubMed2005

Application of RNA interference in tick salivary gland research.

Ticks are obligate ectoparasites that feed on a variety of hosts including mammals, birds and reptiles. Prolonged attachment on the host and an ability to transmit a wide variety of pathogens are the special features of tick feeding. Salivary glands are the major route for secretion of excess fluid, several proteins, and factors that counteract the host immune response and hence play a significant role in the success of tick feeding. RNA interference (RNAi) enables scientists to silence genes encoding proteins in an absolutely sequence specific manner at the mRNA level. This technique has already been successfully employed in analyzing roles of proteins of important functions or in assigning roles to several proteins of unknown functions in a variety of animals. In this review, we outline the process of RNAi and the applicability of RNAi in tick salivary gland research.

Animals↗
PubMed2005

RNA interference and plant parasitic nematodes.

RNA interference (RNAi) has recently been demonstrated in plant parasitic nematodes. It is a potentially powerful investigative tool for the genome-wide identification of gene function that should help improve our understanding of plant parasitic nematodes. RNAi should help identify gene and, hence, protein targets for nematode control strategies. Prospects for novel resistance depend on the plant generating an effective form of double-stranded RNA in the absence of an endogenous target gene without detriment to itself. These RNA molecules must then become available to the nematode and be capable of ingestion via its feeding tube. If these requirements can be met, crop resistance could be achieved by a plant delivering a dsRNA that targets a nematode gene and induces a lethal or highly damaging RNAi effect on the parasite.

Animals↗
PubMed2006

Short interfering RNA (siRNA): tool or therapeutic?

Gene silencing by siRNA (short interfering RNA) is a still developing field in biology and has evolved as a novel post-transcriptional gene silencing strategy with therapeutic potential. With siRNAs, virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening unprecedented opportunities for drug discovery. Besides the well-established role for siRNA as a tool for target screening and validation in vitro, recent progress of siRNA delivery in vivo raised expectations for siRNA drugs as the up-and-coming 'magic bullet'. Whether siRNA compounds will make it as novel chemical entities from 'bench to bedside' will probably depend largely on improving their pharmacokinetics in terms of plasma stability and cellular uptake. Whereas locally administered siRNAs have already entered the first clinical trials, strategies for successful systemic delivery of siRNA are still in a preclinical stage of development. Irrespective of its therapeutic potential, RNAi (RNA interference) has unambiguously become a valuable tool for basic research in biology and thereby it will continue to have a major impact on medical science. In this review, we will give a brief overview about the history and current understanding of RNAi and focus on potential applications, especially as a therapeutic option to treat human disease.

Animals↗