microRNAs associated with the different human Argonaute proteins

MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3′-untranslated region (3′-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1–3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected.     

Argonaute identity defines the length of mature mammalian microRNAs

MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1-4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3' and 5' termini, respectively, as well as the PIWI domain comprising an mRNA 'slicing' activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3' ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.     

Association of Argonaute proteins and microRNAs can occur after cell lysis

MicroRNA (miRNA) target identification is a challenging but important endeavor. Global analyses of the direct mRNA targets of miRNAs have relied heavily upon immunopurification techniques, wherein a core protein component of the miRNA-protein complex, Argonaute (Ago), is immunoprecipitated to isolate associated RNAs. This approach involves the assumption that the selected RNAs were bound to the Ago protein in vivo and that the methodology did not significantly perturb endogenous interactions or produce novel interaction artifacts. To test whether RNAs that coimmunoprecipitate with human Ago were bound in vivo or could associate post-cell lysis, we used an experimental approach that distinguishes between these two origins of interaction. We show that a transfected miRNA mimic, but not a plasmid-expressed miRNA, can interact with human Ago proteins post-lysis. Our results have important implications for the design of miRNP immunoprecipitation experiments.     

Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1‐associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1‐bound miR‐222‐3p, miR‐497‐5p and miR‐21‐5p were significantly higher, and let‐7a‐5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1‐HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF‐related miRNAs compared with the whole serum.     

Redirection of miRNA‐Argonaute Complexes to Specific Target Sites by Synthetic Adaptor Molecules

Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non‐native target sites. Metabolically stable DNA‐LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye‐labeled miRNA model and of specific miRNA‐programmed RISC fractions present in HeLa extracts is demonstrated by pull‐down experiments with biotinylated capture oligonucleotides.     

Functional replacement of Trypanosoma brucei Argonaute by the human slicer Argonaute2

RNA interference (RNAi) is widespread throughout the eukaryotic lineage, from protozoa to man. Central to all RNAi phenomena is a member of the Argonaute protein family, and, in the case of dsRNA-triggered mRNA cleavage, the Ago protein functions as the RNAi endonuclease or slicer. However, at present there is no definite experimental evidence that slicer Argonautes can be interchanged between distantly related organisms. Here, we show that the human slicer Argonaute2 (HsAgo2), but not HsAgo1, functions in RNAi in the early divergent protozoan Trypanosoma brucei, thus mimicking the situation in mammalian cells. This finding indicates that the basic features of the RNAi mechanism are conserved from T. brucei to man.     

Terpyridine Platinum(II) Complexes Inhibit Cysteine Proteases by Binding to Active-site Cysteine

Abstract

Platinum(II) complexes have been demonstrated to form covalent bonds with sulfur-donating ligands (in glutathione, metallothionein and other sulfur-containing biomolecules) or coordination bonds with nitrogen-donating ligands (such as histidine and guanine). To investigate how these compounds interact with cysteine proteases, we chose terpyridine platinum(II) (TP-Pt(II)) complexes as a model system. By using X-ray crystallography, we demonstrated that TP-Pt(II) formed a covalent bond with the catalytic cysteine residue in pyroglutamyl peptidase I. Moreover, by using MALDI (matrix-assisted laser desorption/ionization) and TOF-TOF (time of flight) mass spectrometry, we elucidated that the TP-Pt(II) complex formed a covalent bond with the active-site cysteine residue in two other types of cysteine protease. Taken together, the results unequivocally showed that TP-Pt(II) complexes can selectively bind to the active site of most cysteine proteases. Our findings here can be useful in the design of new anti-cancer, anti-parasite or anti-virus platinum(II) compounds.     

Time Pressure Increases Cooperation in Competitively Framed Social Dilemmas

What makes people willing to pay costs to benefit others? Does such cooperation require effortful self-control, or do automatic, intuitive processes favor cooperation? Time pressure has been shown to increase cooperative behavior in Public Goods Games, implying a predisposition towards cooperation. Consistent with the hypothesis that this predisposition results from the fact that cooperation is typically advantageous outside the lab, it has further been shown that the time pressure effect is undermined by prior experience playing lab games (where selfishness is the more advantageous strategy). Furthermore, a recent study found that time pressure increases cooperation even in a game framed as a competition, suggesting that the time pressure effect is not the result of social norm compliance. Here, we successfully replicate these findings, again observing a positive effect of time pressure on cooperation in a competitively framed game, but not when using the standard cooperative framing. These results suggest that participants'' intuitions favor cooperation rather than norm compliance, and also that simply changing the framing of the Public Goods Game is enough to make it appear novel to participants and thus to restore the time pressure effect.     

Phosphatidic Acid Sequesters Sec18p from cis‐SNARE Complexes to Inhibit Priming

Yeast vacuole fusion requires the activation of cis‐SNARE complexes through priming carried out by Sec18p/N‐ethylmaleimide sensitive factor and Sec17p/α‐SNAP. The association of Sec18p with vacuolar cis‐SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane‐associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA‐rich membrane microdomains before transferring to cis‐SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA‐binding domain blocked Sec18p binding to PA‐liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis‐SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis‐SNAREs and complementation with plasmid‐encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p.      

Argonaute10 as a miRNA locker

The stability and translation efficiency of many messenger RNAs is regulated by microRNAs (miRNAs), which exert their effects through associated Argonaute proteins. In this issue, Zhu, Zhang, and colleagues reveal that plants also exploit miRNA binding by Argonautes as a sequestering mechanism that prevents miRNAs from fulfilling their normal roles.     

siRNA enrichment in Argonaute 2-depleted Blattella germanica

BackgroundRNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation.MethodsUsing the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced.ResultsDepleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals.Conclusions/general significanceThe siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies.     

Argonaute proteins: key players in RNA silencing

During the past decade, small non-coding RNAs have rapidly emerged as important contributors to gene regulation. To carry out their biological functions, these small RNAs require a unique class of proteins called Argonautes. The discovery and our comprehension of this highly conserved protein family is closely linked to the study of RNA-based gene silencing mechanisms. With their functional domains, Argonaute proteins can bind small non-coding RNAs and control protein synthesis, affect messenger RNA stability and even participate in the production of a new class of small RNAs, Piwi-interacting RNAs.     

微小RNA研究进展

MicroRNAs(miRNAs)是一类长度为21-23核苷酸(nt)、进化上比较保守的非编码蛋白质的单链小RNA分子,它们一般通过Dicer酶从具有发夹二级结构的前体RNA加工而来。在真核生物的发育、基因表达等一系列过程中发挥了重要作用。