Monsanto provides technical analysis: Zhang et al. http://bit.ly/xEc4Tt

Technical Analysis: Zhang et al.

Monsanto Company

1/10/2012

Zhang et al. (2011) demonstrate that, among the very large number of microRNAs (miRNAs) in plants, a small number can be detected in human and animal blood. In mice, the authors show that following ingestion of large doses of one particular miRNA (MIR168a), MIR168a was absorbed, was detectable in the serum and liver, protein from a particular gene (LDLRAP1) involved in the removal of LDL (“bad”) cholesterol from blood was reduced and as a result, LDL levels in the mice were increased.

The authors suggest that such a “cross-kingdom” effect – a plant gene product (MIR168a) regulating animal gene expression – may be a common phenomenon; and that miRNAs in food may regulate specific genes in animals based upon matching sequences between plant miRNAs and mammalian genes.

Since this paper was published, Monsanto scientists have thoroughly studied the work and its relevance to the safety assessment of genetically modified (GM) crops and foods derived from them. 

There is too little experimental evidence to conclude that the regulation of animal genes by plant miRNAs is an important diet-mediated phenomenon. MIR168a was relatively abundant in the control chow diet, and yet no effect was seen on LDLRAP1 or LDL cholesterol in mice fed this chow diet.  By contrast, consumption of a large amount of uncooked rice, containing approximately 8-fold higher amounts of MIR168a than the chow diet, resulted in changes in LDLRAP1 and, consequently, an increase in serum LDL cholesterol.  It is noteworthy that mice were fed the human equivalent of over 73 poundsof cooked rice per person per day, which exceeds the highest rice-consuming population (97.5^th percentile group) by 80 to 100 fold.  The fact that rural Chinese populations consuming high-rice diets have been shown to have lower levels of LDL than their urban counterparts (with lower rice intake) would suggest that the activity of MIR168a in humans is negligible or at least is sufficiently small that other environmental determinants are more important in regulating  LDL concentration than  MIR168a. In short, the relevance of dietary concentrations of MIR168a or other miRNAs in humans remains to be established.  

It is important to remember that humans regularly consume plants that contain small RNAs.  Recent research by Ivashuta et al. (2009, Food and Chemical Toxicology 47:353–360) demonstrated that many existing plant RNA’s share sequences with human genes. Further, humans regularly consume animal derived foods with mammalian miRNAs with 100% identity to human genes.  Despite the routine ingestion of plant and animal small RNAs, no impacts on human gene regulation or health have been reported.  Treatment of disease via oral ingestion of RNA-based medications has not been accomplished despite more than a decade of effort by the pharmaceutical industry.   Systemic suppression of specific target genes in humans has not been possible with oral administration of small RNAs, even when using RNA constructs specifically designed to achieve gene suppression and when employing modified RNAs to enhance stability. 

This is an important area of research, but given what is known about the ubiquitous nature of RNA in all whole foods and about the unsuccessful efforts to develop oral RNA pharmaceutical products, much more information is needed before it can be concluded that dietary miRNAs regularly have any meaningful impact on mammalian or human gene regulation.

Importantly, the authors state: “It is unlikely that such high concentrations of mature plant miRNAs can be achieved in serum, plasma, and organs of humans or animals via food intake.” Based on the available information, the results with the abundant MIR168a are not sufficient to support a broad conclusion  that plant miRNAs present in food are part of a common and general mechanism for “cross-kingdom” regulation animal genes.

• After a careful examination of the paper, we have identified a number of relevant facts that should be taken into account when looking at data and the relevancy of the findings. 
• Of the many thousands of plant miRNAs, only a small number are found in human or animal blood.
• The absence of most plant miRNAs in serum indicates:
  • Absorption may be selective;
  • Only some miRNAs in foods have properties which allow them to survive in foods, the GI tract, and serum;
  • Only relatively abundant miRNAs are present at high enough levels to be detected;
  • Or some combination of these factors.
• MIR168a is among the more abundant miRNAs in many plants, but even allowing for this MIR168a appears to be disproportionately found in animal tissues and is also considerably more stable in rice than MIR156a or MIR166a (see Table S3 of Zhang et al.)  This suggests that for some reason MIR168a is preferentially absorbed and/or preserved (before or after absorption) relative to other miRNAs.
• Changes in MIR168a levels with concomitant changes in LDLRAP1 expression and LDL cholesterol were observed following higher doses (raw rice diet) in mice; however no effect on LDLRAP1 or LDL cholesterol was seen when animals were fed an ordinary chow diet.  Consequently, there may well be little or no effect on mice eating a more diverse, ordinary diet.
• The findings with MIR168a may represent a rare or unique case, resulting from the uncharacteristically high abundance of MIR168a in rice and disproportionate absorption and/or preservation of MIR168a in combination with the high homology (gene sequence match) to LDLRAP1.
• The loss of MIR168a effects occurred with less than a 10-fold reduction in diet concentration, indicating that this phenomenon is highly dose-dependant.  The ability to observe this phenomenon may be related to the high-dosing regimens employed.
• The authors cannot exclude the possibility that in-vivo LDLRAP1 reduction and concomitant LDL increase were due to radical changes in diet.  In addition to higher levels of MIR168a, the rice-only diet was higher in carbohydrates and deficient in fats and proteins, and the animals were in a starvation-state.
• While high doses of MIR168a influence cholesterol levels in mice, the relevance of dietary intakes of MIR168a or other miRNAs in the human diet remains to be established.
• The fact that rural Chinese populations consuming high-rice diets have been shown to have lower levels of LDL than their urban counterparts (who have lower rice intake) suggests that the activity of MIR168a in humans is negligible or at least is sufficiently small that other environmental determinants of LDL concentration may overcome the effects of MIR168a on cholesterol.

There is a broad foundation of evidence that supports the safety of GM crops that express siRNAs.  These data have been reviewed and accepted by Regulatory authorities globally.  Monsanto will continue to examine all new evidence published in the scientific literature and our own studies.  We are committed to the safety of our products and to safety of the food and feed products produced from them.

Link to Cell Research: http://bit.ly/wwls3w

Ingested plant miRNAs regulate gene expression in animals

 Hervé Vaucheret and Yves Chupeau  

Institut Jean-Pierre Bourgin, INRA, 78000 Versailles, France

The incidence of genetic material or epigenetic information transferred from one organism to another is an important biological question. A recent study demonstrated that plant small RNAs acquired orally through food intake directly influence gene expression in animals after migration through the plasma and delivery to specific organs.

Non-protein coding RNAs, and in particular small RNAs, were recently revealed as master chief regulators of gene expression in all organisms. Endogenous small RNAs come in different flavors, depending on their mode of biogenesis. Most microRNAs (miRNA) and short interferring RNAs (siRNA) derive from long double-stranded RNA (dsRNA) precursors that are processed into small RNA duplexes, 20 to 25-nt long, by RNaseIII enzymes called Dicer ¹. One strand of small RNA duplexes is loaded onto an Argonaute protein that executes silencing by cleaving or repressing the translation of homologous mRNA ². In certain species, RNA cleavage is followed by DNA methylation and/or histone modification, leading to heritable epigenetic modification ³.

Endogenous small RNAs play essential roles during development and stress responses, and control transposable elements and chromatin states. Small RNAs can also be produced in response to invasion by exogenous nucleic acids from viruses, bacteria, transgenes, etc ⁴. Under these circumstances, small RNAs act in defense mechanisms by directing the destruction of the invader. Importantly, exogenous small RNAs and some endogenous small RNAs are mobile within certain organisms ⁵. Moreover, exogenous small RNAs can be amplified during the defense mechanisms, allowing the spreading of RNA silencing from the cell where it is activated to the rest of the organism ⁶.

Immediately after its discovery, the potent effect of small RNAs has been exploited to specifically downregulate gene expression in a timely controlled manner. Such technology is commonly used in the laboratory but also for biomedical applications. Indeed, artificially synthesized small RNAs or dsRNA can be introduced exogenously to look for transient and localized effects. Artificial small RNAs can also be expressed durably from stably integrated transgenes or using replicating viruses. In certain organisms, a simple and convenient manner to induce RNA silencing consists in feeding the recipient host with bacteria expressing dsRNA homologous to the cognate target. This method works with worms and paramecia ^7, 8. Small RNAs or dsRNAs can also be transferred from plant to pests such as insects that eat leaves or nematodes that infect roots. Indeed, transgenic plants expressing dsRNA homologous to essential genes of insect pests or nematodes specifically resist these parasites due to the silencing activity of small RNAs expressed from the plant transgenes ^9, 10, 11.

Given that human food relies largely on plants, it was expected that researchers would look for the outcome of plant small RNAs after food intake, and for a potential effect of the ingested plant small RNAs on the expression of human genes. In the recent paper by Zhang et al. ¹² in Cell Research, cloning and sequencing of small RNAs in human serum revealed that plant miRNAs represented about 5% of mammalian miRNAs. Plant miRNAs are 2′-O-methyl modified at their 3′ end, which renders them resistant to periodate, whereas human miRNAs have free 2′ and 3′ hydroxyl, which renders them sensitive to periodate. The plant miRNAs cloned from human serum were resistant to periodate, indicating that they are genuine plant miRNAs, probably coming from the food intake. Confirming this hypothesis, the concentration of plant miRNAs was higher in the serum of rice-fed mice compared with chow diet-fed mice. Moreover, adding plant miRNAs to chow diet resulted in an increase of plant miRNA concentration in mouse serum. Interestingly, cooking did not impair the accumulation of plant miRNAs, indicating that they are resistant to heat and thus could be acquired from both raw and cooked meals.

Plant miRNAs were primarily detected in plasma microvesicles (MV) ¹². MVs are shed from almost all cell types and have the potential to selectively interact with specific target cells and mediate intercellular communication by transporting lipids, RNA and proteins. Consistently, plant miRNAs were detected in various tissues, including liver, intestine and lung. Different plant miRNAs accumulated at different levels, which also varied from one tissue to another, but their levels could reach up to one tenth of the most abundant human miRNA. Indeed, human miRNAs have a dynamic range of accumulation, extending from 1 copy to > 10 000 copies per cell. The amount of the most abundant plant miRNA found in human serum, miR168, was 3.2 × 10⁻⁶ fmol (1 920 copies) per 100 pg of total RNA, equivalent to 850 copies per cell, which is equivalent to the average amount of a human miRNA.

Plant miR168 is one of the most important miRNA in plants. Indeed, it regulatesAGO1 mRNA, which encodes the core component of the plant RNA silencing complex. As such, AGO1 binds to the vast majority of plant miRNAs. RegulatingAGO1 by miR168 ensures a feedback loop that allows adjusting the level of AGO1 to the amount of miRNAs in the cell. AGO1 also binds to exogenous siRNAs induced by defense mechanisms. Exogenous siRNAs competes with miRNAs and deplace the AGO1/miR168 equilibrium to adjust the amount of AGO1 to the amount of exogenous siRNAs required for the plant defenses ¹³.

Unexpectedly, plant miR168 exhibits a high degree of complementarity with the exon 4 of mammalian LDLRAP1 (Low Densitiy Lipoprotein Receptor Adapter Protein 1) mRNA ¹². LDLRAP1 is a liver-enriched mRNA encoding a protein that facilitates the removal of LDL (Low Density Lipoprotein) from the circulatory system. The level of LDLRAP1 protein but not LDLRAP1 mRNA was decreased in rice-fed mice compared with chow diet-fed mice, indicating that plant miR168 executes silencing like a human miRNA in human cells. The effect of miR168 was sequence specific because adding the LDLRAP1 complementarity sequence to a luciferase reporter resulted in a decrease of luciferase activity in cells co-transfected with miR168. In addition, mutating the LDLRAP1 complementarity sequence abolished the silencing effect of miR168.

The authors of this study hypothesized that epithelial cells in the intestine might take up miRNAs in food, package them into MVs and release them into the circulatory sytem ¹². The secreted MVs could then deliver exogenous plant miRNA to target organs where they could regulate cognate mRNAs. They tested this hypothesis by transfecting human intestinal epithelial Caco-2 cells with plant miR168, and used the MVs released by the Caco-2 cells to treat HepG2 cells. Immunoprecipitation revealed that miR168 and LDLRAP1 mRNA associate with human RNA silencing complex in HepG2 cells treated with Caco-2 MVs transfected with miR168. Eventually, LDLRAP1 protein level was decreased in HepG2 cells, indicating that MVs carried a functional miR168 to specific organs.

Downregulation of LDLRAP1 in the liver causes decreased endocytosis of LDL by liver cells and impairs removal of LDL from the plasma ¹². Consistent with the silencing effect of plant miR168 on LDLRAP1 protein, LDL levels in mouse plasma were elevated following miR168 uptake. This effect was specific to miR168 because injection of an anti-miR168 oligonucleotide blocked the increase in LDL levels.

These results raise the question of whether food-derived small RNAs could play an active role in human/animal health. This question is valid for both plant miRNAs and animal miRNAs. Indeed, this study also revealed that adding an animal miRNA, miR-150, to the mouse diet, allows the increase of miR-150 levels in the liver and downregulation of its natural target c-Myb ¹². Human diets are extremely diverse and rely on various amounts and various species of plants and animals in different parts of the world. Whether physiological and/or pathological differences induced by the foods could be partly determined by the small RNA repertoire of each diet remains to be determined.

Plants encode hundreds of thousands of different small RNAs ¹⁴. Given that six nucleotides of perfect complementary between the “seed” region of a small RNA and its target is sufficient to promote RNA silencing in mammals ¹⁵, how many plant miRNAs have the potential to actually regulate gene expression in animals? One could speculate that biologically active plant miRNAs in our diet are orders of magnitude lower than biologically active animal miRNAs that are ingested when eating meat. This issue will certainly be addressed in the near future. Given the stability of miRNAs in the gut, their influence on the bacterial community is yet another field of interrogation.

During the last century, the outcome of proteins has been examined, mostly due to the extension of bovine spongiform encephalopathy, a disease caused by prions (a class of degenerative proteins), which raised important concerns in animal farming and human nutrition. After the release of genetically modified organisms (GMO) on the market, the fate of trangenes and transgene products in the digestive tract have also been questioned. With this study, the possible incidence of RNA contained in the food diet on animal/human health will certainly become an explosive field of investigation. This study also implies precise awareness from biotechnologists who intend to make use of dsRNA, especially in the field of plant protection against pests.

Food and Chemical Toxicology

journal homepage:  www.elsevier.com/ locate/foodchemtox

Article history:

Received 8 July 2008

Accepted 14 November 2008

doi:10.1016/j.fct.2008.11.025

Endogenous small RNAs in grain: Semi-quantification and sequence

homology to human and animal genes

Sergey I. Ivashuta, Jay S. Petrick *, Sara E. Heisel, Yuanji Zhang, Liang Guo, Tracey L. Reynolds,

James F. Rice, Edwards Allen, James K. Roberts

Monsanto Company, 800 N. Lindbergh Blvd., Mail Code O3F, St. Louis, MO 63167, USA

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.

link to NSMB: http://bit.ly/woHp8N

The N domain of Argonaute drives duplex unwinding during RISC assembly  

Pieter Bas Kwak & Yukihide Tomari

Small RNAs, such as microRNAs and small interfering RNAs, act through Argonaute (Ago) proteins as a part of RNA-induced silencing complexes (RISCs). To make RISCs, Ago proteins bind and subsequently unwind small RNA duplexes, finally leaving one strand stably incorporated. Here we identified the N domain of human AGO2 as the initiator of duplex unwinding during RISC assembly. We discovered that a functional N domain is strictly required for small RNA duplex unwinding but not for precedent duplex loading or subsequent target cleavage. We postulate that RISC assembly is tripartite, comprising (i) RISC loading, whereby Ago undergoes conformational opening and loads a small RNA duplex, forming pre-RISC; (ii) wedging, whereby the end of the duplex is pried open through active wedging by the N domain, in preparation for unwinding; and (iii) unwinding, whereby the passenger strand is removed through slicer-dependent or slicer-independent unwinding, forming mature RISC.

linkt to Nucleic Acids Research: http://bit.ly/s8tlQ5

Expression determinants of mammalian argonaute proteins in mediating gene silencing

RNA interference occurs by two main processes: mRNA site-specific cleavage and non-cleavage-based mRNA degradation or translational repression. Site-specific cleavage is carried out by argonaute-2 (Ago2), while all four mammalian argonaute proteins (Ago1–Ago4) can carry out non-cleavage-mediated inhibition, suggesting that Ago1, Ago3 and Ago4 may have similar but potentially redundant functions. It has been observed that in mammalian tissues, expression of Ago3 and Ago4 is dramatically lower compared with Ago1; however, an optimization of the Ago3 and Ago4 coding sequences to include only the most common codon at each amino acid position was able to augment the expression of Ago3 and Ago4 to levels comparable to that of Ago1 and Ago2. Thus, we examined whether particular sequence features exist in the coding region of Ago3 and Ago4 that may prevent a high level of expression. Swapping specific sub-regions of wild-type and optimized Ago sequence identified the portion of the coding region (nucleotides 1–1163 for Ago-3 and 1–1494 for Ago-4) that is most influential for expression. This finding has implications for the evolutionary conservation of Ago proteins in the mammalian lineage and the biological role that potentially redundant Ago proteins may have.

Cell: http://bit.ly/tQirzD

Conserved Function of lincRNAs in Vertebrate Embryonic Development despite Rapid Sequence Evolution

Igor Ulitsky^{1, 2, 3, 5}, Alena Shkumatava^{1, 2, 3, 5}, Calvin H. Jan^{1, 2, 3, 4}, Hazel Sive^1, 3 and David P. Bartel^{1, 2, 3, ,}  

¹ Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
² Howard Hughes Medical Institute
³ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Corresponding author

⁴ Present address: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco and California Institute for Quantitative Biosciences, San Francisco, CA 94158, USA

⁵ These authors contributed equally to this work

• Highlights

► The zebrafish genome encodes hundreds of long intervening noncoding RNAs (lincRNAs) ► Only 29 of 567 lincRNAs have detectable sequence homology with mammalian lincRNAs ► Two lincRNAs, 

• Summary

• Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.

Link to Cell: http://bit.ly/tty5Ae

ceRNAs: miRNA Target Mimic Mimics

Ignacio Rubio-Somoza¹, Detlef Weigel^1, ,  , José-Manuel Franco-Zorilla², Juan Antonio García² and Javier Paz-Ares^2, ,  

¹ Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany
² Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain

Corresponding author

Corresponding author

MAIN TEXT

In a recent issue of Cell, four papers described regulatory interactions among messenger RNAs (mRNAs) that share target sequences for the same microRNAs (miRNAs) (Cesana et al., 2011,Karreth et al., 2011,Sumazin et al., 2011,Tay et al., 2011). The authors state that these mRNAs, dubbed “competing endogenous RNAs (ceRNAs),” define a new layer of regulation of miRNA activity that has only recently been discovered. It may be new for animals and humans, but the principle of this phenomenon had been identified a while ago in plants, where it is known as “target mimicry.”

Plant biologists not only played a central role in defining the function of small RNAs in gene silencing, but they also made important contributions to our understanding of miRNA action. In both plants and animals, miRNAs negatively affect their targets through a variety of transcriptional and posttranscriptional mechanisms. In addition, the biogenesis and activity of miRNAs themselves can be regulated at several levels. One of these is through target mimicry. In 2007, we reported that the IPS1 (INDUCED BY PHOSPHATE STARVATION1) RNA altered the protein levels of PHO2(PHOSPHATE2) by modulating the effects of miR399 on the stability and translation of PHO2 mRNA (Franco-Zorrilla et al., 2007). The RNAs of both IPS1and PHO2 have highly conserved sequence motifs complementary to miR399, with IPS1 having additional bases that interrupt the position where miR399 would normally guide cleavage of its target.

A series of experiments supported a model in which noncleavable IPS1 sequesters miR399 and thus prevents it from inhibiting PHO2 mRNA accumulation and translation. We coined the term “target mimicry” for this endogenous regulatory mechanism of miRNA activity. Although IPS1 encodes a noncoding RNA, these early findings already pointed to the possibility that noncleavable target sites in plants could perhaps not only act in trans, but also in cis, as found to be the case in animals. We subsequently confirmed that artificial target mimic sites in plants could indeed reduce translation efficiency in cis (Todesco et al., 2010).

A few weeks after natural and artificial target mimics had been described for plants (Franco-Zorrilla et al., 2007), artificial target mimics were introduced as “decoy targets” for miRNAs in animals (Ebert et al., 2007). Subsequently, a theoretical paper suggested that many natural miRNA target sites could act as miRNA decoys or target mimics in animals. In such a scenario, changes in the expression of some miRNA targets (or mimics) would alter the ability of a miRNA to reduce the activity of other targets (Seitz, 2009).

Last year, Pandolfi and colleagues reported an exciting discovery of a naturally occurring noncoding RNA produced by a pseudogene that acts as a natural target mimic in human tumors (Poliseno et al., 2010). A recent Essay in Cell (Salmena et al., 2011) summarized the conclusions from this and some of the prior work, including our initial discovery of “target mimics” (Franco-Zorrilla et al., 2007) and the theoretical prediction by Seitz, 2009. The Essay formally enumerated the many ways in which noncoding and coding RNAs with similar miRNA target sites could affect each others' activity. The authors renamed such RNAs with shared and competing target sites as ceRNAs. They also stated that their theory “challenged the notion that a protein-coding mRNA must be translated into a protein to exert function,” although there has been ample prior evidence for noncoding functions of protein-coding mRNAs in animals and plants. Such examples include mRNAs that produce small RNAs because they form natural antisense transcripts or are routed through the trans-acting siRNA pathway.

In the September 30, 2011 issue Cell, three groups described new ceRNAs in animal and human cells (Cesana et al., 2011,Karreth et al., 2011,Sumazin et al., 2011,Tay et al., 2011). The new work goes substantially beyond what was known before and is the kind of research that one is pleased to read about in the pages of Cell. However, the impact of these papers would not have been lessened if they had acknowledged that the path for these findings was paved in plants. In our opinion, at the core of these articles is the target mimicry principle, which endows nondegradable miRNA targets with regulatory potential.

Please give credit where credit is due. Plant biologists would rightfully be ridiculed if they claimed to have made new discoveries while equivalent phenomena were already known from animals or fungi. Given that the value of the world's agriculture is more than three times that of the entire pharmaceutical industry and that many more people die each year of hunger and malnutrition than from cancer, it is time that scientists of all stripes paid more attention to plant biology.

RNA Journal: http://bit.ly/sgXc2l

A role for transcription from a piRNA cluster in de novo piRNA production

Molecular Cell: http://bit.ly/rJCf5h

The 3′-to-5′ Exoribonuclease Nibbler Shapes the 3′ Ends of MicroRNAs Bound to Drosophila Argonaute1

EMBO Journal: http://bit.ly/vhKN7n

PABP is not essential for microRNA-mediated translational repression and deadenylation in vitro

The EMBO Journal 30, 4998 - 5009 (25 November 2011) |doi:10.1038/emboj.2011.426

Takashi Fukaya and Yukihide Tomari

MicroRNAs silence their complementary target genes via formation of the RNA-induced silencing complex (RISC) that contains an Argonaute (Ago) protein at its core. It was previously proposed that GW182, an Ago-associating protein, directly binds to poly(A)-binding protein (PABP) and interferes with its function, leading to silencing of the target mRNAs. Here we show that Drosophila Ago1-RISC induces silencing via two independent pathways: shortening of the poly(A) tail and pure repression of translation. Our data suggest that although PABP generally modulates poly(A) length and translation efficiency, neither PABP function nor GW182–PABP interaction is a prerequisite for these two silencing pathways. Instead, we propose that each of the multiple functional domains within GW182 has a potential for silencing, and yet they need to act together in the context of full-length GW182 to exert maximal silencing.