The RNA-Induced Silencing Complex Is a Mg2+-Dependent Endonuclease

AbstractIn the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5′ cleavage products are readily detected for RNAi in vitro, only 3′ cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5′ and 3′ cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5′ and 3′ cleavage products in vitro; cleavage requires Mg2+, but not Ca2+, and the cleavage product termini suggest a role for Mg2+ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn2+, but not by Ca2+ or Mg2+. We propose that during catalysis, a Mg2+ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified

Reconstitution of human RNA interference in budding yeast

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism

Network development in biological gels: role in lymphatic vessel development

In this paper, we present a model that explains the prepatterning of lymphatic vessel morphology in collagen gels. This model is derived using the theory of two phase rubber material due to Flory and coworkers and it consists of two coupled fourth order partial differential equations describing the evolution of the collagen volume fraction, and the evolution of the proton concentration in a collagen implant; as described in experiments of Boardman and Swartz (Circ. Res. 92, 801–808, 2003). Using linear stability analysis, we find that above a critical level of proton concentration, spatial patterns form due to small perturbations in the initially uniform steady state. Using a long wavelength reduction, we can reduce the two coupled partial differential equations to one fourth order equation that is very similar to the Cahn–Hilliard equation; however, it has more complex nonlinearities and degeneracies. We present the results of numerical simulations and discuss the biological implications of our model

Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments

One-year risk of stroke after transient ischemic attack or minor stroke in Hunter New England, Australia (INSIST Study)

Background: One-year risk of stroke in transient ischemic attack and minor stroke (TIAMS) managed in secondary care settings has been reported as 5–8%. However, evidence for the outcomes of TIAMS in community care settings is limited. Methods: The INternational comparison of Systems of care and patient outcomes In minor Stroke and TIA (INSIST) study was a prospective inception cohort community-based study of patients of 16 general practices in the Hunter–Manning region (New South Wales, Australia). Possible-TIAMS patients were recruited from 2012 to 2016 and followed-up for 12 months post-index event. Adjudication as TIAMS or TIAMS-mimics was by an expert panel. We established 7-days, 90-days, and 1-year risk of stroke, TIA, myocardial infarction (MI), coronary or carotid revascularization procedure and death; and medications use at 24 h post-index event. Results: Of 613 participants (mean age; 70 ± 12 years), 298 (49%) were adjudicated as TIAMS. TIAMS-group participants had ischemic strokes at 7-days, 90-days, and 1-year, at Kaplan-Meier (KM) rates of 1% (95% confidence interval; 0.3, 3.1), 2.1% (0.9, 4.6), and 3.2% (1.7, 6.1), respectively, compared to 0.3, 0.3, and 0.6% of TIAMS-mimic-group participants. At one year, TIAMS-group-participants had twenty-five TIA events (KM rate: 8.8%), two MI events (0.6%), four coronary revascularizations (1.5%), eleven carotid revascularizations (3.9%), and three deaths (1.1%), compared to 1.6, 0.6, 1.0, 0.3, and 0.6% of TIAMS-mimic-group participants. Of 167 TIAMS-group participants who commenced or received enhanced therapies, 95 (57%) were treated within 24 h post-index event. For TIAMS-group participants who commenced or received enhanced therapies, time from symptom onset to treatment was median 9.5 h [IQR 1.8–89.9]. Conclusion: One-year risk of stroke in TIAMS participants was lower than reported in previous studies. Early implementation of antiplatelet/anticoagulant therapies may have contributed to the low stroke recurrence

The characteristics of patients with possible Transient Ischemic Attack and Minor Stroke in the Hunter and Manning Valley regions, Australia (the INSIST Study)

This is the final version. Available on open access from the American Academy of Neurology via the DOI in this record. Background: Transient ischemic attack (TIA) and minor stroke (TIAMS) are risk factors for stroke recurrence. Some TIAMS may be preventable by appropriate primary prevention. We aimed to recruit “possible-TIAMS” patients in the INternational comparison of Systems of care and patient outcomes In minor Stroke and TIA (INSIST) study. Methods: A prospective inception cohort study performed across 16 Hunter–Manning region, Australia, general practices in the catchment of one secondary-care acute neurovascular clinic. Possible-TIAMS patients were recruited from August 2012 to August 2016. We describe the baseline demographics, risk factors and pre-event medications of participating patients. Results: There were 613 participants (mean age; 69 ± 12 years, 335 women), and 604 (99%) were Caucasian. Hypertension was the most common risk factor (69%) followed by hyperlipidemia (52%), diabetes mellitus (17%), atrial fibrillation (AF) (17%), prior TIA (13%) or stroke (10%). Eighty-nine (36%) of the 249 participants taking antiplatelet therapy had no known history of cardiovascular morbidity. Of 102 participants with known AF, 91 (89%) had a CHA2DS2-VASc score ≥ 2 but only 47 (46%) were taking anticoagulation therapy. Among 304 participants taking an antiplatelet or anticoagulant agent, 30 (10%) had stopped taking these in the month prior to the index event. Conclusion: This study provides the first contemporary data on TIAMS or TIAMS-mimics in Australia. Community and health provider education is required to address the under-use of anticoagulation therapy in patients with known AF, possibly inappropriate use of antiplatelet therapy and possibly inappropriate discontinuation of antiplatelet or anticoagulation therapy.National Health and Medical Research Counci

Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed

West Nile virus encodes a microRNA-like small RNA in the 3′ untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3′ untranslated region (3′-UTR) of the flavivirus genome, in particular the terminal 3′ stem–loop (3′SL) fulfils multiple functions in virus replication and virus–host interactions. Using the Kunjin strain of WNV (WNVKUN), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3′SL. Transcription of WNVKUN pre-miRNA (3′SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNVKUN virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation