The influence of the 5 '-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site

Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecule

The RNAi-Competent Malaria Parasite: A Novel Strategy to Knock Down Plasmodium Genes via Non-Canonical RNAi

Malaria, caused by apicomplexan parasites of the Plasmodium species, is one of the deadliest infectious diseases worldwide. Despite the urgent need to identify new drug targets and vaccine candidates, a large proportion of the Plasmodium genes are uncharacterized, as tools to study gene function are limited. In many eukaryotes, genes can be silenced via RNA interference (RNAi) using artificial short hairpin RNAs (shRNAs). However, Plasmodium parasites lack the machinery required for RNAi. In this study, I therefore engineered a non-canonical RNAi machinery into the rodent parasite Plasmodium berghei (P. berghei). To this end, I exploited a non-canonical RNAi pathway which requires only a single protein, Argonaute 2 (Ago2), and a specifically designed shRNA, a so-called AgoshRNA, for gene silencing. I generated a P. berghei line constitutively expressing Ago2, named PbAgo2, and demonstrated that this parasite can complete its life cycle through the mammalian and insect host, despite exhibiting a reduced growth in blood and mosquito stages. Expression of AgoshRNAs targeting the mRNA of the green fluorescent protein GFP (constitutively expressed by PbAgo2) induced a potent knockdown of GFP both in blood and in non-erythrocytic stages. As different AgoshRNAs mediated gene silencing to various levels, target gene expression could be fine-tuned. AgoshRNA-mediated gene knockdown was also possible for endogenous genes, and the knockdown of a non-essential gene phenocopied the full knockout. Additionally, the expression of a blood-stage-essential gene was reduced using RNAi. The analysis of the transcriptome of PbAgo2 by RNA sequencing suggested a possible interaction between Ago2 and a Plasmodium mRNA storage protein as a putative reason for the growth impairment. To further increase the potential applications of the RNAi-competent parasite, Ago2 expression was restricted to the liver stage using a stage-specific promoter. This transgenic line behavee indistinguishable from wild type and the expression of an AgoshRNA targeting GFP silenced fluorescence exclusively in late liver stages. In summary, PbAgo2 is a potent tool to modulate gene expression without the need to alter the genetic locus. In contrast to existing tools, PbAgo2 provides the option to target genes exclusively in a single life cycle stage, to multiplex different AgoshRNAs enabling the simultaneous knockdown of multiple genes, or to screen for phenotypes using a library of AgoshRNAs. This novel, RNAi-competent parasite line opens a wealth of new options to annotate genes in Plasmodium

Expression of anti-viral shRNA molecules in transgenic zebrafish

 Development of transgenic zebrafish specifically resistant to Viral Hemorrhagic Septicemia Virus mediated by short-hairpin RNA interference. Introduction of shRNAs by Tol2 transgenisis in to zebrafish overwhelmed multiple facets of the endogenous microRNA pathway including Exportin-5 and Argonaute-2 and prevented normal zebrafish development

Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism. The shRNA precursor is processed by Dicer into small interfering RNAs (siRNAs) and subsequently programs the RNAi-induced silencing complex (RISC) to find a complementary target mRNA (mRNA) for post-transcriptional gene silencing. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer to be processed directly by the Ago2 nuclease of the RISC complex. We named this design AgoshRNA as these molecules depend on Ago2 both for processing and subsequent silencing activity. This alternative AgoshRNA processing route yields only a single active RNA strand, an important feature to restrict off-target effects induced by the passenger strand of regular shRNAs. It is therefore important to understand this novel AgoshRNA processing route in mechanistic detail such that one can design the most effective and selective RNA reagents. We performed a systematic analysis of the optimal base pair (bp) composition at the top and bottom of AgoshRNA molecules. In this study, we document the importance of the 5' end nucleotide (nt) and a bottom mismatch. The optimized AgoshRNA design exhibits improved RNAi activity across cell types. These results have important implications for the future design of more specific RNAi therapeutic

Silencing of HIV-1 by AgoshRNA molecules

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by the expression of a short hairpin RNA (shRNA). shRNA molecules enter the RNAi pathway at the Dicer processing step. Recent studies indicated that the cellular microRNA miR-451 is not recognized by Dicer, but that it is processed instead by the Argonaute 2 (Ago2) protein. Subsequently, Dicer-independent shRNAs were described that rely on Ago2 for processing, as well as the subsequent silencing step. We called these AgoshRNA molecules because they depend on Ago2 both for maturation and activation. Processing of an AgoshRNA yields only a single active RNA strand, thus reducing the chance of adverse off-target effects induced by the passenger strand of regular shRNAs. In this study, we converted several anti-HIV-1 shRNAs into AgoshRNAs. Seven of the 21 designed AgoshRNAs were potent anti-HIV molecules, although their RNAi activity is generally somewhat reduced compared with the matching shRNAs. The AgoshRNA candidates revealed no cellular toxicity. This may relate to the absence of passenger strand expression, which was verified for these AgoshRNA candidates. Furthermore, we demonstrate that a toxic shRNA can be converted into a non-toxic AgoshRN

Towards Antiviral shRNAs Based on the AgoshRNA Design

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these "AgoshRNA" molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxi

Boosting AgoshRNA activity by optimized 5’-terminal nucleotide selection

RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5ʹ-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5ʹ-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes

Novel AgoshRNA molecules for silencing of the CCR5 co-receptor for HIV-1 infection.

Allogeneic transplantation of blood stem cells from a CCR5-Δ32 homozygous donor to an HIV-infected individual, the "Berlin patient", led to a cure. Since then there has been a search for approaches that mimic this intervention in a gene therapy setting. RNA interference (RNAi) has evolved as a powerful tool to regulate gene expression in a sequence-specific manner and can be used to inactivate the CCR5 mRNA. Short hairpin RNA (shRNA) molecules can impair CCR5 expression, but these molecules may cause unintended side effects and they will not be processed in cells that lack Dicer, such as monocytes. Dicer-independent RNAi pathways have opened opportunities for new AgoshRNA designs that rely exclusively on Ago2 for maturation. Furthermore, AgoshRNA processing yields a single active guide RNA, thus reducing off-target effects. In this study, we tested different AgoshRNA designs against CCR5. We selected AgoshRNAs that potently downregulated CCR5 expression on human T cells and peripheral blood mononuclear cells (PBMC) and that had no apparent adverse effect on T cell development as assessed in a competitive cell growth assay. CCR5 knockdown significantly protected T cells from CCR5 tropic HIV-1 infection