Interaction between a J-domain co-chaperone and a specific Argonaute protein contributes to microRNA function in animals.

MicroRNAs (miRNAs) are essential regulators of several biological processes. They are loaded onto Argonaute (AGO) proteins to achieve their repressive function, forming the microRNA-Induced Silencing Complex known as miRISC. While several AGO proteins are expressed in plants and animals, it is still unclear why specific AGOs are strictly binding miRNAs. Here, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans. DNJ-12 does not interact with ALG-2, the other major miRNA-specific AGO, and PRG-1 and RDE-1, two AGOs involved in other small RNA pathways, making it a specific actor in ALG-1-dependent miRNA-mediated gene silencing. The loss of DNJ-12 causes developmental defects associated with defective miRNA function. Using the Auxin Inducible Degron system, a powerful tool to acutely degrade proteins in specific tissues, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to the decrease of several miRNAs and prevents their loading onto ALG-1. This study uncovers the importance of a co-chaperone for the miRNA function in vivo and provides insights to explain how different small RNAs associate with specific AGO in animals.

Defining the contribution of microRNA-specific Argonautes with slicer capability in animals.

microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicer residues in the canonical microRNA pathway is still unclear in animals. To address this, we created Caenorhabditis elegans strains with mutated slicer residues in the endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the mutation in ALG-1 and ALG-2 catalytic residues affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the slicer residues of ALG-1 and ALG-2 contribute differentially to regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the catalytic tetrad of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicer residues of miRNA-specific Argonautes contribute to maintaining levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

Defining the contribution of microRNA-specific slicing Argonautes in animals.

microRNAs regulate gene expression through interaction with an Argonaute protein family member. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicing activity in the canonical microRNA pathway is still unclear in animals. To address the importance of slicing Argonautes in animals, we created Caenorhabditis elegans strains, carrying catalytically dead endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the loss of ALG-1 and ALG-2 slicing activity affects overall animal fitness and causes phenotypes, reminiscent of miRNA defects, only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the catalytic activity of ALG-1 and ALG-2 differentially regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the slicing activity of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicing activity of miRNA-specific Argonautes function to maintain the levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

Casein kinase 1 and 2 phosphorylate Argonaute proteins to regulate miRNA-mediated gene silencing.

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.

CSDE1 promotes miR-451 biogenesis.

MicroRNAs are sequentially processed by RNase III enzymes Drosha and Dicer. miR-451 is a highly conserved miRNA in vertebrates which bypasses Dicer processing and instead relies on AGO2 for its maturation. miR-451 is highly expressed in erythrocytes and regulates the differentiation of erythroblasts into mature red blood cells. However, the mechanistic details underlying miR-451 biogenesis in erythrocytes remains obscure. Here, we report that the RNA binding protein CSDE1 which is required for the development of erythroblasts into erythrocytes, controls the expression of miR-451 in erythroleukemia cells. CSDE1 binds miR-451 and regulates AGO2 processing of pre-miR-451 through its N-terminal domains. CSDE1 further interacts with PARN and promotes the trimming of intermediate miR-451 to the mature length. Together, our results demonstrate that CSDE1 promotes biogenesis of miR-451 in erythroid progenitors.

A specific type of Argonaute phosphorylation regulates binding to microRNAs during C. elegans development.

Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.

Regulation and different functions of the animal microRNA-induced silencing complex.

Among the different types of small RNAs, microRNAs (miRNAs) are key players in controlling gene expression at the mRNA level. To be active, they must associate with an Argonaute protein to form the miRNA induced silencing complex (miRISC) and binds to specific mRNA through complementarity sequences. The miRISC binding to an mRNA can lead to multiple outcomes, the most frequent being inhibition of the translation and/or deadenylation followed by decapping and mRNA decay. In the last years, several studies described different mechanisms modulating miRISC functions in animals. For instance, the regulation of the Argonaute protein through post-translational modifications can change the miRISC gene regulatory activity as well as modulate its binding to proteins, mRNA targets and miRISC stability. Furthermore, the presence of RNA binding proteins and multiple miRISCs at the targeted mRNA 3' untranslated region (3'UTR) can also affect its function through cooperation or competition mechanisms, underlying the importance of the 3'UTR environment in miRNA-mediated repression. Another way to regulate the miRISC function is by modulation of its interactors, forming different types of miRNA silencing complexes that affect gene regulation differently. It is also reported that the subcellular localization of several components of the miRNA pathway can modulate miRISC function, suggesting an important role for vesicular trafficking in the regulation of this essential silencing complex. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3'UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.

CSDE1 attenuates microRNA-mediated silencing of PMEPA1 in melanoma.

MicroRNAs and RNA-binding proteins (RBPs) primarily target the 3' UTR of mRNAs to control their translation and stability. However, their co-regulatory effects on specific mRNAs in physiology and disease are yet to be fully explored. CSDE1 is an RBP that promotes metastasis in melanoma and mechanisms underlying its oncogenic activities need to be completely defined. Here we report that CSDE1 interacts with specific miRNA-induced silencing complexes (miRISC) in melanoma. We find an association of CSDE1 with AGO2, the essential component of miRISC, which is facilitated by target mRNAs and depends on the first cold shock domain of CSDE1. Both CSDE1 and AGO2 bind to 3' UTR of PMEPA1. CSDE1 counters AGO2 binding, leading to an increase of PMEPA1 expression. We also identify a miRNA, miR-129-5p, that represses PMEPA1 expression in melanoma. Collectively, our results show that PMEPA1 promotes tumorigenic traits and that CSDE1 along with miR-129-5p/AGO2 miRISC act antagonistically to fine-tune PMEPA1 expression toward the progression of melanoma.

CSDE1 controls gene expression through the miRNA-mediated decay machinery.

In animals, miRNAs are the most prevalent small non-coding RNA molecules controlling posttranscriptional gene regulation. The Argonaute proteins (AGO) mediate miRNA-guided gene silencing by recruiting multiple factors involved in translational repression, deadenylation, and decapping. Here, we report that CSDE1, an RNA-binding protein linked to stem cell maintenance and metastasis in cancer, interacts with AGO2 within miRNA-induced silencing complex and mediates gene silencing through its N-terminal domains. We show that CSDE1 interacts with LSM14A, a constituent of P-body assembly and further associates to the DCP1-DCP2 decapping complex, suggesting that CSDE1 could promote the decay of miRNA-induced silencing complex-targeted mRNAs. Together, our findings uncover a hitherto unknown mechanism used by CSDE1 in the control of gene expression mediated by the miRNA pathway.

Defend Thyself and Thy Offspring.

Three recent studies (Dodson and Kennedy, 2019; Lev et al., 2019; Ouyang et al., 2019) reveal that germ granule formation is necessary to protect germline-expressed genes from improper small RNA-mediated silencing. Loss of this protection leads to accumulation of small RNAs, impacting gene expression in multiple subsequent generations.

A guide to microRNA-mediated gene silencing.

As an important regulator of gene expression in eukaryotes, microRNAs (miRNAs) have been subject to extensive research to understand their pleiotropic roles in development and pathological processes. Here, we review strategies that have been developed to map microRNA-target interactions and the most effective methods to attribute post-transcriptional gene regulation through miRNA-mediated silencing. We further discuss the importance of the biological context for silencing.

Somatic and Germline MicroRNAs Form Distinct Silencing Complexes to Regulate Their Target mRNAs Differently.

Animal germ cells possess a specific post-transcriptional regulatory context allowing the storage of maternal transcripts in the oocyte until their translation at a specific point in early development. As key regulators of gene expression, miRNAs repress translation mainly through mRNA destabilization. Thus, germline miRNAs likely use distinct ways to regulate their targets. Here, we use C. elegans to compare miRNA function within germline and somatic tissues. We show that the same miRNA displays tissue-specific gene regulatory mechanisms. While translational repression occurs in both tissues, targeted mRNAs are instead stabilized in the germline. Comparative analyses of miRNA silencing complexes (miRISC) demonstrate that their composition differs from germline to soma. We show that germline miRNA targets preferentially localize to perinuclear regions adjacent to P granules, and their repression is dependent on the core P granule component GLH-1. Together, our findings reveal the existence of different miRISC in animals that affect targeted mRNAs distinctively.

Endothelial microRNAs regulating the NF-κB pathway and cell adhesion molecules during inflammation.

The surface of endothelial cells is covered with cell adhesion molecules, including E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM- 1) , that mediate the adhesion and extravasation of leukocytes and play pivotal roles in inflammatory response. microRNAs (miRNAs) regulate the expression of these important cell adhesion molecules through two distinct major mechanisms, namely via modulating the proinflammatory NF-κB pathway, which controls their transcription, and via directly targeting them. The present review highlights the role of various miRNAs in controlling the expression of E-selectin, ICAM-1, and VCAM-1: a type of regulation that can be harnessed for therapeutic prevention of inflammation-associated diseases such as atherosclerosis and sepsis. The roles of secreted miRNAs as paracrine regulators, and cell adhesion molecule-based miRNA delivery are also addressed.-Zhong, L., Simard, M. J., Huot, J. Endothelial microRNAs regulating the NF-κB pathway and cell adhesion molecules during inflammation.

[Biogenesis of small non-coding RNAs in animals]. / La biogenèse des ARN courts non codants chez les animaux.

In recent years, the discovery of small non-coding RNAs has opened an all new field in molecular biology. Indeed, these non-coding sequences give rise to powerful regulators of gene expression. Nowadays, different types of small non-coding RNAs have been described. Of these, the best-characterized types are microRNAs, piRNAs (Piwi-interacting RNAs) and siRNAs (small interfering RNAs). Because of their fine-tuning important function in the regulation of gene and genome expression, an aberrant expression level of those small non-coding RNAs are associated to several pathologies. While this new research field is attracting attention, many aspects to be discovered. This review focuses on the biogenesis pathways of microRNAs, piRNAs and siRNAs in animals.

p38 activation induces production of miR-146a and miR-31 to repress E-selectin expression and inhibit transendothelial migration of colon cancer cells.

Extravasation of circulating cancer cells determines their metastatic potential. This process is initiated by the adhesion of cancer cells to vascular endothelial cells through specific interactions between endothelial adhesion receptors such as E-selectin and their ligands on cancer cells. In the present study, we show that miR-146a and miR-181b impede the expression of E-selectin by repressing the activity of its transcription factor NF-κB, thereby impairing the metastatic potentials of colon cancer cells by decreasing their adhesion to, and migration through, the endothelium. Among the two microRNAs, only miR-146a is activated by IL-1ß, through the activation of p38, ERK and JNK MAP kinases, as well as their downstream transcription factors GATA2, c-Fos and c-Jun. Inhibiting p38 MAP kinase increases NF-κB activity, at least partially via miR-146a. Inhibiting p38 also increases the expression of E-selectin at the post-transcriptional level via decreasing miR-31, which targets E-selectin mRNA and also depends on p38 for its expression. In response to IL-1ß, p38 MAP kinase hence represses the expression of E-selectin at the transcriptional and the post-transcriptional levels, via miR-146a and miR-31, respectively. These results highlight novel mechanisms by which p38 downregulates the expression of E-selectin through different microRNAs following inflammatory stimuli associated to cancer progression.

The R148.3 Gene Modulates Caenorhabditis elegans Lifespan and Fat Metabolism.

Despite many advances, the molecular links between energy metabolism and longevity are not well understood. Here, we have used the nematode model Caenorhabditis elegans to study the role of the yet-uncharacterized gene R148.3 in fat accumulation and lifespan. In wild-type worms, a R148.3p::GFP reporter showed enhanced expression throughout life in the pharynx, in neurons, and in muscles. Functionally, a protein fusing a predicted 22 amino acid N-terminal signal sequence (SS) of R148.3 to mCherry displayed robust accumulation in coelomyocytes, indicating that R148.3 is a secreted protein. Systematic depletion of R148.3 by RNA interference (RNAi) at L1 but not at young-adult stage enhanced triglyceride accumulation, which was associated with increased food uptake and lower expression of genes involved in lipid oxidation. However, RNAi of R148.3 at both L1 and young-adult stages robustly diminished mean and maximal lifespan of wild-type worms, and also abolished the long-lived phenotypes of eat-2 and daf-2/InsR mutants. Based on these data, we propose that R148.3 is an SS that modulates fat mass and longevity in an independent manner.

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo.

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function in vivo Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.

TEG-1 CD2BP2 controls miRNA levels by regulating miRISC stability in C. elegans and human cells.

MiRNAs post-transcriptionally regulate gene expression by recruiting the miRNA-induced silencing complex (miRISC) to target mRNAs. However, the mechanisms by which miRISC components are maintained at appropriate levels for proper function are largely unknown. Here, we demonstrate that Caenorhabditis elegans TEG-1 regulates the stability of two miRISC effectors, VIG-1 and ALG-1, which in turn affects the abundance of miRNAs in various families. We demonstrate that TEG-1 physically interacts with VIG-1, and complexes with mature let-7 miRNA. Also, loss of teg-1 in vivo phenocopies heterochronic defects observed in let-7 mutants, suggesting the association of TEG-1 with miRISC is necessary for let-7 to function properly during development. Loss of TEG-1 function also affects the abundance and function of other microRNAs, suggesting that TEG-1's role is not specific to let-7. We further demonstrate that the human orthologs of TEG-1, VIG-1 and ALG-1 (CD2BP2, SERBP1/PAI-RBP1 and AGO2) are found in a complex in HeLa cells, and knockdown of CD2BP2 results in reduced miRNA levels; therefore, TEG-1's role in affecting miRNA levels and function is likely conserved. Together, these data demonstrate that TEG-1 CD2BP2 stabilizes miRISC and mature miRNAs, maintaining them at levels necessary to properly regulate target gene expression.

p38 and JNK pathways control E-selectin-dependent extravasation of colon cancer cells by modulating miR-31 transcription.

Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to vascular endothelial cells. It requires the interaction between adhesion receptors such as E-selectin present on endothelial cells and their ligands on cancer cells. Notably, E-selectin influences the metastatic potential of breast, bladder, gastric, pancreatic, and colorectal carcinoma as well as of leukemia and lymphoma. Here, we show that E-selectin expression induced by the pro-inflammatory cytokine IL-1ß is directly and negatively regulated by miR-31. The transcription of miR-31 is activated by IL-1ß. This activation depends on p38 and JNK MAP kinases, and their downstream transcription factors GATA2, c-Fos and c-Jun. The miR-31-mediated repression of E-selectin impairs the metastatic potential of colon cancer cells by decreasing their adhesion to, and migration through, the endothelium. These results highlight for the first time that microRNA mediates E-selectin-dependent extravasation of colon cancer cells.