Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization.

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

MiR-652-3p promotes malignancy and metastasis of cancer cells via inhibiting TNRC6A in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) was one of the most prevalent life-threatening cancers. Metastasis is the leading cause of cancer-related death in HCC. MiRNAs play essential roles in cancer metastasis. METHODS: Expression of miR-652-3p in HCC was assessed. Function experiments of miR-652-3p and trinucleotide repeat-containing gene 6A protein (TNRC6A) were performed both in vitro and in vivo. mRNA sequencing, PCR, and western blot were performed to verify the target genes and pathway of miR-652-3p. The lung metastasis and xenograft cancer model in nude mice was established to investigate the effects of the miR-652-3p and TRNC6A on tumor metastasis in vivo. The relationship between the expression of the miR-652-3p, TNRC6A and the prognosis of HCC patients was analyzed. RESULTS: Upregulated miR-652-3p was found in the tumor tissues of HCC, especially in metastatic HCC patients. Overexpression of miR-652-3p promoted and knockdown of miR-652-3p suppressed HCC metastasis both in vitro and in vivo. MiR-652-3p promoted HCC metastasis via regulating the EMT pathway. TNRC6A was identified as a direct target of miR-652-3p, and the knockdown of TNRC6A restored repressed EMT and HCC metastasis caused by the inhibition of miR-652-3p. Clinical results revealed that high expression of miR-652-3p and low expression of TNRC6A were positively correlated to shortened overall survival and disease-free survival in HCC patients. CONCLUSIONS: MiR-652-3p promotes EMT and HCC metastasis by inhibiting TNRC6A expression in HCC. MiR-652-3p and TNRC6A may serve as potential biomarkers to predict prognosis in HCC patients with metastasis.

N-terminal Ago-binding domain of GW182 contains a tryptophan-rich region that confer binding to the CCR4-NOT complex.

GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex.

LINC01082 Inhibits Non-Small Cell Lung Cancer by Targeting the miR-543/TNRC6A Axis.

Non-small cell lung cancer (NSCLC) accounts for over 80% of lung cancer cases and have poor clinical outcomes. Increasing number of lncRNAs are reported to be implicated in the carcinogenesis of NSCLC. Previous lncRNA-seq results showed that LINC01082 was under-expressed in several cancer types. In the current study, we focused on the role of LINC01082 in NSCLC development. An online bioinformatics tool was utilized to assess the expression profile of LINC01082, miR-543, and TNRC6A in NSCLC samples. RT-qPCR analysis was performed for evaluating LINC01082, TNRC6A and miR-543 expression in cells (NSCLC cells vs. normal lung cells). Impact of LINC01082 upregulation on cell proliferation in vitro was investigated by MTT and EdU experiments. Transwell assay was applied to analyze the migration and invasion of NSCLC cells. The cell apoptosis after plasmid transfection was detected by flow cytometry. The interactions among LINC01082, miR-543 and TNRC6A were measured by RNA pulldown and luciferase reporter assays. We showed that LINC01082 levels were downregulated in NSCLC samples and NSCLC cells. Overexpression of LINC01082 inhibited NSCLC cell proliferation, migration and invasion and strengthened cell apoptosis. LINC01082 directly bound to miR-543, and miR-543 targeted TNRC6A. TNRC6A was downregulated and miR-543 was overexpressed in NSCLC cells. miR-543 inhibition suppressed malignant cellular behaviors. TNRC6A knockdown reversed the effects of LINC01082 on the malignant character of NSCLC cells. In conclusion, LINC01082 exerts an antioncogenic role in NSCLC via interaction with miR-543 to regulate TNRC6A expression.

lncRNA AK017368 promotes proliferation and suppresses differentiation of myoblasts in skeletal muscle development by attenuating the function of miR-30c.

Long noncoding RNAs (lncRNAs) have been reported to play diverse roles in biologic and pathologic processes, including myogenesis. We found that lncRNA AK017368 is highly expressed in skeletal muscle cells. Functional analyses showed that overexpression of AK017368 promoted proliferation and restrained differentiation of myoblasts; whereas inhibition of AK017368 had completely opposite effects in vitro In mice, knockdown of AK017368 promoted muscle hypertrophy in vivo RNA molecules of AK017368 acted mechanistically as competing endogenous RNAs to target micro-RNA (miR)-30c, which was supported by the results of bioinformatics analyses and dual-luciferase reporter assays. It has been shown that lncRNA AK017368 competes with trinucleotide repeat containing-6A (Tnrc6a) for miR-30c. Tnrc6a was previously reported to promote proliferation and inhibit differentiation of myoblast cells, whereas miR-30c targets the 3'-UTR of Tnrc6a mRNA to weaken its function. Taken together, lncRNA AK017368 promotes proliferation and inhibits differentiation of myoblast cells by attenuating function of miR-30c.-Liang, T., Zhou, B., Shi, L., Wang, H., Chu, Q., Xu, F., Li, Y., Chen, R., Shen, C., Schinckel, A. P. lncRNA AK017368 promotes proliferation and suppresses differentiation of myoblasts in skeletal muscle development by attenuating the function of miR-30c.

High expression of miR-30c-5p in satellite cells of high-fat diet-induced obese rabbits inhibited satellite cell proliferation and promoted differentiation.

It was revealed in our previous study that the expression of miR-30c-5p in the skeletal muscle of rabbits fed high-fat diet was highly expressed. In the present study, we further investigated the function of miR-30c-5p in proliferation and differentiation of skeletal muscle satellite cell (SMSC). The results obtained in the present study showed that the skeletal muscle fibers of the rabbits fed the standard normal diet (SND) were orderly, regular, and uniform after HE staining, however, the muscle fibers of the rabbits fed the high-fat diet (HFD) were generally atrophied, some were arranged disorderly, the intercellular space was enlarged, the nucleus was increased, and the morphology and position were abnormal. Compared with the SND group, it was observed that the weekly weight gain and fat percentage were relatively higher, and also the levels of the serum biochemical indexes such as glucose, cholesterol, and triglyceride increased significantly in the rabbits fed with HFD. In addition, the results after the transfection of miR-30c-5p mimic, mimic NC (negative control), miR-30c-5p inhibitor, and inhibitor NC into the SMSCs showed that the cell counting kit-8 (CCK-8) proliferation experiment suggested that the number of cells in the over expression group was significantly lower than that in the mimic NC group at 48, 72, 96 h of cell proliferation. At 48, 72, 120 h, the number of cells in the inhibitor group was significantly higher than that in the mimic NC group. The number of EdU positive cells decreased significantly in the over expression group compared with the mimic NC group, however, it increased significantly in the inhibitor group compared with the inhibitor NC group. Moreover, compared with the mimic NC group, the myotube area increased significantly in the miR-30c-5p mimic group, whereas it decreased significantly in the miR-30c-5p inhibitor group as compared with the inhibitor NC group. In addition, we found that trinucleotide repeat containing adaptor 6A (TNRC6A) was successfully validated as a target gene for miR-30c-5p. The expression of TNRC6A in the miR-30c-5p mimic group was significantly lower than that in the mimic NC group. It was further observed that the expression of TNRC6A increased significantly in the miR-30c-5p inhibitor group as compared to that in the inhibitor NC group. Taken together, the results obtained in this study showed that miR-30c-5p promotes the differentiation as well as inhibits the proliferation of rabbit skeletal muscle satellite cells, and TNRC6A is a target gene of miR-30c-5p.

Hypoxic BMSC-derived exosomal miR-652-3p promotes proliferation and metastasis of hepatocarcinoma cancer cells via targeting TNRC6A.

Cancer microenvironment plays an important role in the proliferation and metastasis of hepatocarcinoma cancer cells (HCC). Exosomes from bone marrow-derived mesenchymal stem cells (BMSCs) are a component of the cancer microenvironment. In this study, we reveal that miRNA-652-3P from BMSC-derived exosomes promotes proliferation and metastasis in HCC. The ability of cancer proliferation, migration and invasion can be evaluated after co-culture by CCK-8, wound healing and transwell assay. Isolated exosomes were identified by transmission electron microscopy (TEM) and the biomarkers of the purified exosomes were showed in West-blotting (WB). MiR-652-3p was detected in the HepG2 and 7721 after co-culturing with exosome derived from BMSCs under different conditions. Target authentication was performed by a luciferase reporter assay to confirm the presumptive target of miR-652-3p. After overexpressing miR-652-3p, the mRNA and protein expression level of TNRC6A in HCC was examined by q-PCR and WB. Further, we observed greater miR-652-3p upregulation in hypoxic BMSCs-exosomes than in normal- exosomes. In addition, a miR-652-3p inhibitor attenuates the proliferation and metastasis of HCC cells after co-culturing with BMSCs. Our data demonstrate that hypoxic BMSCs-derived exosomal miR-652-3p promotes proliferation in HCC cells by inhibiting TNRC6A. The BMSCs-derived exosomal miR-652-3p may help find patient-targeted therapies in hepatocarcinoma cancer.

Identification of Phosphorylated Amino Acids in Human TNRC6A C-Terminal Region and Their Effects on the Interaction with the CCR4-NOT Complex.

Human GW182 family proteins have Argonaute (AGO)-binding domains in their N-terminal regions and silencing domains, which interact with RNA silencing-related proteins, in their C-terminal regions. Thus, they function as scaffold proteins between the AGO protein and RNA silencing-related proteins, such as carbon catabolite repressor4-negative on TATA (CCR4-NOT) or poly(A)-binding protein (PABP). Our mass spectrometry analysis and the phosphorylation data registered in PhosphoSitePlus, a post-translational modification database, suggested that the C-terminal region of a human GW182 family protein, TNRC6A, has at least four possible phosphorylation sites, which are located near the region interacting with the CCR4-NOT complex. Among them, two serine residues at amino acid positions 1332 and 1346 (S1332 and S1346) were certainly phosphorylated in human HeLa cells, but other two serine residues (S1616 and S1691) were not phosphorylated. Furthermore, it was revealed that the phosphorylation patterns of TNRC6A affect the interaction with the CCR4-NOT complex. When S1332 and S1346 were dephosphorylated, the interactions of TNRC6A with the CCR4-NOT complex were enhanced, and when S1616 and S1691 were phosphorylated, such interaction was suppressed. Thus, phosphorylation of TNRC6A was considered to regulate the interaction with RNA silencing-related factors that may affect RNA silencing activity.

Aberrant expression of TNRC6a and miR-21 during myocardial infarction.

In the present study, aberrant expression of trinucleotide repeat-containing gene 6a (TNRC6a) and miR-21 was noted and documented in rat myocardial infarction. Briefly, Sprague-Dawley rat model was used for the development of myocardial infarction. Experiments such as histological analysis were carried out to confirm the histopathology of the myocardial infarction. The expression profile of TNRC6a and miR-21 was identified by using quantitative real-time PCR. In addition, immunoblotting was performed to validate the expression profile of TNRC6a and phosphatase and tensin homolog (PTEN). The histological analysis confirmed the progress of myocardial infarction in rat model. As the disease progresses, the protein TNRC6a expresses abnormally which in turn up-regulates the miR-21 after 3rd and 5th week of infarction. Interestingly, miR-21 binds with its specific target genes PTEN and thereby degrades the target mRNA; as a result, its expression was down-regulated progressively and paved the development of myocardial infarction. The present study concludes that the aberrant expression of TNRC6a and miR-21 was documented during myocardial infarction. These findings play an important role in the diagnosis as well as pave a way for the development of drug targets for treating myocardial infarction.

Comprehensive Identification of Nuclear and Cytoplasmic TNRC6A-Associating Proteins.

Trinucleotide repeat-containing gene 6A protein (TNRC6A) is an essential protein for microRNA-mediated gene silencing. TNRC6A functions in the cytoplasm as a platform protein interacting with Argonaute protein, on which microRNA is loaded for RNA silencing, and decapping enzymes or deadenylation protein complexes to induce mRNA degradation. We previously revealed that TNRC6A shuttles between the cytoplasm and nucleus. However, the function of TNRC6A in the nucleus is unclear. Here, we performed a comprehensive identification of the nuclear and cytoplasmic interacting proteins of TNRC6A protein by mass spectrometry and identified multiple proteins involved in the nuclear and cytoplasmic complexes. We found that many RNA degradation pathway proteins were involved in both nuclear and cytoplasmic TNRC6A complexes, suggesting that RNA silencing may occur via TNRC6A in both nucleus and cytoplasm or that they were involved in other important function in the nucleus. Furthermore, proteins identified in the nuclear TNRC6A complex were categorized into the spliceosomal pathway. This may mean that TNRC6A regulates splicing in the nucleus. In contrast, pathogen infection- and RNA transport-associated proteins were identified in the cytoplasmic TNRC6A complex. Thus, TNRC6A may be also involved in these pathways in the cytoplasm.

MicroRNAs recruit eIF4E2 to repress translation of target mRNAs.

MicroRNAs (miRNAs) recruit the RNA-induced silencing complex (RISC) to repress the translation of target mRNAs. While the 5' 7-methylguanosine cap of target mRNAs has been well known to be important for miRNA repression, the underlying mechanism is not clear. Here we show that TNRC6A interacts with eIF4E2, a homologue of eIF4E that can bind to the cap but cannot interact with eIF4G to initiate translation, to inhibit the translation of target mRNAs. Downregulation of eIF4E2 relieved miRNA repression of reporter expression. Moreover, eIF4E2 downregulation increased the protein levels of endogenous IMP1, PTEN and PDCD4, whose expression are repressed by endogenous miRNAs. We further provide evidence showing that miRNA enhances eIF4E2 association with the target mRNA. We propose that miRNAs recruit eIF4E2 to compete with eIF4E to repress mRNA translation.

Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins.

As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

miR-26a-5p Regulates TNRC6A Expression and Facilitates Theca Cell Proliferation in Chicken Ovarian Follicles.

Ovarian theca cells play an indispensable role in ovarian follicular development and hormone secretion. miR-26a-5p was reported to be differentially expressed in mature and immature chicken ovaries in our previous study; however, the role of miR-26a-5p in regulating ovarian follicle function is still unclear. In this study, we demonstrated that the expression dynamics of TNRC6A mRNA in either chicken ovaries or follicles showed an opposite trend compared with that of chicken miR-26a-5p expression. miR-26a-5p inhibited TNRC6A mRNA expression by directly targeting its 3'-untranslated region in cultured chicken theca cells. Overexpression of miR-26a-5p promoted chicken follicular theca cell proliferation in vitro. Furthermore, overexpression of miR-26a-5p and knockdown of TNRC6A significantly upregulated the antiapoptotic BCL-2 gene. Taken together, this study revealed the expression dynamics of miR-26a-5p and TNRC6A in chicken ovaries and ovarian follicles and the relationship between the expression of miR-26a-5p and TNRC6A in chicken ovarian theca cells. These results suggest that miR-26a-5p facilitates chicken ovarian theca cell proliferation by targeting the TNRC6A gene.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.