A pinboard by
Sybil Wong, PhD

Completed PhD in Biochemistry in 2015 with a focus on microRNA-mediated gene silencing


The role of key protein families Ago & GW182 in miRNA action

Latest update, 5 July 2017:

Argonaute (AGO) proteins mediate posttranscriptional gene silencing through formation of the microRNA-induced silencing complex (miRISC). Bridge et al. identify LIM-domain-containing proteins as essential for miRISC formation through a phosphorylation-dependent mechanism. This is critical for posttranscriptional gene silencing and reveals that miRISC functionality is maintained by ‘‘AGO switching.’’


Perinatal protein malnutrition alters expression of miRNA biogenesis genes Xpo5 and Ago2 in mice brain.

Abstract: Due to its widespread incidence, maternal malnutrition remains one of the major non-genetic factors affecting the development of newborn's brain. While all nutrients have certain influence on brain maturation, proteins appear to be the most critical for the development of neurological functions. An increasing number of studies point out that the effects of early-life nutritional inadequacy has long lasting effects on the brain and lead to permanent deficits in learning and behavior. Epigenetic mechanisms provide a potential link between the nutrition status during critical periods and changes in gene expression that may lead to disease phenotypes. Among those epigenetic mechanisms microRNAs (miRNAs) emerge as promising molecules for the link between nutrition and gene expression due to their relevance in many central nervous system functions. The objective of the current study was to evaluate the impact of perinatal protein malnutrition on the development of male and female mice offspring and to analyze the expression of the genes involved in the miRNA biogenesis pathway in different mouse brain structures. We demonstrated that early nutritional stress such as exposition to a protein-deficient diet during gestation and lactation reduced the hippocampal weight, delayed offspring's development and deregulated the expression of Xpo5 and Ago2 genes in hippocampus and hypothalamus of weanling mice. Moreover, an overall increase in mature miRNAs was consistent with the induction of Xpo5 mRNA. Altered miRNA biogenesis could modify the availability and functionality of miRNA becoming a causal factor of the adverse effects of protein malnutrition.

Pub.: 17 Mar '17, Pinned: 22 Apr '17

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

Abstract: Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.

Pub.: 22 Jan '17, Pinned: 26 Jan '17

GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

Abstract: MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.

Pub.: 10 Dec '16, Pinned: 20 Dec '16

Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons.

Abstract: MicroRNAs (miRNAs) are small regulatory RNAs that regulate gene expression posttranscriptionally by base pairing to the target mRNAs in animal cells.KRas, an oncogene known to be repressed by let-7a miRNAs, is expressed and needed for the differentiation of mammalian sympathetic neurons and PC12 cells. We documented a loss of let-7a activity during this differentiation process without any significant change in the cellular level of let-7a miRNA. However, the level of Ago2, an essential component that is associated with miRNAs to form RNP-specific miRNA (miRNP) complexes, shows an increase with neuronal differentiation. In this study, differentiation-induced phosphorylation and the subsequent loss of miRNA from Ago2 were noted, and these accounted for the loss of miRNA activity in differentiating neurons. Neuronal differentiation induces the phosphorylation of mitogen-activated protein kinase p38 and the downstream kinase mitogen- and stress-activated protein kinase 1 (MSK1). This in turn upregulates the phosphorylation of Ago2 and ensures the dissociation of miRNA from Ago2 in neuronal cells. MSK1-mediated miRNP inactivation is a prerequisite for the differentiation of neuronal cells, where let-7a miRNA gets unloaded from Ago2 to ensure the upregulation ofKRas, a target of let-7a. We noted that the inactivation of let-7a is both necessary and sufficient for the differentiation of sympathetic neurons.

Pub.: 10 Feb '16, Pinned: 08 Sep '16

The role of TP53 in miRNA loading onto AGO2 and in remodelling the miRNA-mRNA interaction network.

Abstract: DNA damage transactivates tumour protein p53 (TP53)-regulated surveillance, crucial in suppressing tumorigenesis. TP53 mediates this process directly by transcriptionally modulating gene and microRNA (miRNA) expression and indirectly by regulating miRNA biogenesis. However, the role of TP53 in regulating miRNA-AGO2 loading and global changes in AGO2 binding to its gene targets in response to DNA damage are unknown. These processes might be novel mechanisms by which TP53 regulates miRNAs in response to DNA damage.To show the network of miRNA-mRNA interactions that occur in response to DNA damage, we stimulated TP53 wild-type and null cell-lines with doxorubicin and performed RNA sequencing from total RNA (RNA-Seq) and AGO2-immunoprecipitated RNA (AGO2-RIP-Seq). We used a combined AGO2 RIP-seq and AGO2 PAR-CLIP-seq (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) approach to determine the exact sites of interaction between the AGO2-bound miRNAs and their mRNA targets.TP53 directly associated with AGO2, and induced and reduced loading of a subset of miRNAs, including the lethal 7 (let-7) miRNA family members, onto AGO2 in response to DNA damage. Although mutated TP53 maintained its capacity to interact with AGO2, it mediated unloading instead of loading of let-7 family miRNAs, thereby reducing their activity. We determined the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence and absence of TP53. Furthermore, we showed that miRNAs whose cellular abundance or differential loading onto AGO2 was regulated by TP53 were involved in an intricate network of regulatory feedback and feedforward circuits that fine-tuned gene expression levels in response to DNA damage to permit the repair of DNA damage or initiation of programmed cell death.Control of AGO2 loading by TP53 is a new mechanism of miRNA regulation in carcinogenesis.UK Medical Research Council, Action Against Cancer.

Pub.: 28 Aug '15, Pinned: 08 Sep '16

Early origin and adaptive evolution of the GW182 protein family, the key component of RNA silencing in animals.

Abstract: The GW182 proteins are a key component of the miRNA-dependent post-transcriptional silencing pathway in animals. They function as scaffold proteins to mediate the interaction of Argonaute (AGO)-containing complexes with cytoplasmic poly(A)-binding proteins (PABP) and PAN2-PAN3 and CCR4-NOT deadenylases. The AGO-GW182 complexes mediate silencing of the target mRNA through induction of translational repression and/or mRNA degradation. Although the GW182 proteins are a subject of extensive experimental research in the recent years, very little is known about their origin and evolution. Here, based on complex functional annotation and phylogenetic analyses, we reveal 448 members of the GW182 protein family from the earliest animals to humans. Our results indicate that a single-copy GW182/TNRC6C progenitor gene arose with the emergence of multicellularity and it multiplied in the last common ancestor of vertebrates in 2 rounds of whole genome duplication (WGD) resulting in 3 genes. Before the divergence of vertebrates, both the AGO- and CCR4-NOT-binding regions of GW182s showed significant acceleration in the accumulation of amino acid changes, suggesting functional adaptation toward higher specificity to the molecules of the silencing complex. We conclude that the silencing ability of the GW182 proteins improves with higher position in the taxonomic classification and increasing complexity of the organism. The first reconstruction of the molecular journey of GW182 proteins from the ancestral metazoan protein to the current mammalian configuration provides new insight into development of the miRNA-dependent post-transcriptional silencing pathway in animals.

Pub.: 25 Jun '15, Pinned: 08 Sep '16

Mechanistic insights on the Dicer-independent AGO2-mediated processing of AgoshRNAs.

Abstract: Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.

Pub.: 01 Apr '15, Pinned: 08 Sep '16

Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates.

Abstract: When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3' untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response.

Pub.: 05 Jul '13, Pinned: 08 Sep '16

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2.

Abstract: MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Pub.: 03 May '13, Pinned: 08 Sep '16