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MicroRNA 4651 regulates nonsense-mediated mRNA decay by targeting SMG9 mRNA
Gene 701 (2019) 65–71
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Research paper
MicroRNA 4651 regulates nonsense-mediated mRNA decay by targeting SMG9 mRNA ⁎
Yanjie Tana, Zhenfa Maa, Yi Jina, Ruojun Zonga, Jian Wuc, , Zhuqing Rena,b,
T
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a Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China b The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China c College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-4651 SMG9 NMD
Nonsense-mediated mRNA decay (NMD) is originally identified as a conserved RNA surveillance mechanism that rapidly degrades aberrant mRNA containing premature termination codons (PTCs). However, the molecular regulation mechanisms by which microRNAs inhibit NMD has not been well understood. Here we identified that miR-4651 participated in the NMD pathway by downregulating expression levels of SMG9. We provided evidences that (1) Overexpression of miR-4651 mimic significantly inhibited the expression of SMG9 (P < 0.05); (2) NMD substrates genes, TBL2 and GADD45B were both increased at mRNA and protein expression levels when SMG9 was suppressed by siRNA, whereas decreased by SMG9 overexpression; (3) Expression of SMG9 was significantly increased but TBL2, GADD45B were significantly decreased when cells transfected with miR-4651 inhibitor (P < 0.05). These results indicated that miR-4651 regulated NMD by targeting SMG9 mRNA. Our study highlights that miR-4651 represses NMD. miR-4651 targets SMG9 and represses the expression levels of SMG9. NMD activity is decreased additionally when SMG9 is inhibited. The present study provides evidence for microRNA/NMD regulatory mechanism.
1. Introduction Eukaryotic gene expression involves a complex series of biological processes. From transcription of genetic information to protein synthesis, post-transcriptional modification and remodeling are essential for the translation of ribosomes into mature mRNAs (Fernà ndez et al., 2011). Several monitoring mechanisms ensure the fidelity and accuracy of these processes. Nonsense mediated mRNA decay (NMD) is a conservative surveillance mechanism of transcripts quality that recognizes and degrades aberrant transcripts containing a premature termination codons (PTC) (Chang et al., 2007; Popp and Maquat, 2013; Lykkeandersen and Bennett, 2014; Karousis et al., 2016). A quarter of PTCs are produced by mutations in human genetic diseases, e.g. cancers often exhibit abnormal transcription and mRNA processing. Therefore, NMD can protect cells from the accumulation of potentially harmful Cterminal truncated proteins encoded by PTC-containing mRNAs (Yamashita et al., 2009). In addition to DNA mutations, an error in transcription, or inefficiently spliced pre-mRNAs can also lead to
abnormal transcripts with PTC (Feng et al., 2017). NMD was also found to be responsible for degradation of 3–10% of normal transcripts in human, therefore serving as an widespread gene regulatory mechanism (Peccarelli and Kebaara, 2014). The machinery of NMD consists of the factors, UPF1, UPF2, UPF3, SMG1, SMG8 and SMG9 involved in PTC recognition, and SMG5, SMG6, SMG7 participating in the degradation of the target mRNAs (Schmidt et al., 2015). The presence of “surveillance complex”, discriminates PTC-containing mRNAs from normal mRNAs and triggers degradation of abnormal transcripts. A “surveillance complex” termed “SURF” was first assembled before the termination of translation. SURF includes SMG1, UPF1, eRF1, and eRF3 (Schmidt et al., 2015). If the exon junction complex (EJC) is located > 50–55 nucleotides downstream of the stop codon, the SURF and EJC interactions ensure that the termination code is identified as a PTC (Schmidt et al., 2015). This “judgment” followed by SMG1 forms the SMG1 complex (SMG1C) with SMG8 and SMG9 to phosphorylate UPF1. It is the single essential event to trigger all the later processes leading to the degradation of an mRNA (Melero
Abbreviations: SMG9, nonsense mediated mRNA decay associated PI3K related kinase9; NMD, nonsense mediated mRNA decay; UPF1, Up-framshift 1; PTC, premature termination codon; SMG1C, SMG1 complex; IDR, intrinsically disordered region; EJC, exon junction complex ⁎ Corresponding authors at: College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. E-mail addresses: [email protected] (J. Wu), [email protected] (Z. Ren). https://doi.org/10.1016/j.gene.2019.03.031 Received 24 December 2018; Received in revised form 11 March 2019; Accepted 17 March 2019 Available online 19 March 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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2.2. Cell culture
et al., 2014). SMG9 can inhibit SMG1 kinase activity in the isolated SMG1C, indicating that they regulate SMG1 during remodeling of the surveillance complex (Fernà ndez et al., 2011). SMG9 is a 520-amino acid protein that contains a nucleotide triphosphatase domain in the center. SMG9 could regulate the generation of SMG1C. Its N-terminal is a 180-residue internal unordered region (IDR) followed by a well-folded C-terminal domain, both of domains have the ability binding to SMG1 and stabilizing the integrity of the SMG1C (Fernà ndez et al., 2011). In humans and nematodes, SMG9 protein affects the stability of PTC-containing mRNA (Yamashita et al., 2009). Cells cannot recognize transcripts containing PTC with loss-offunction mutations of SMG9 (Shaheen et al., 2016). Patients with SMG9 deficiency exhibit extensive transcriptional disorders, suggesting that SMG9 plays an important role in post-transcriptional regulation and surveillance (Fernà ndez et al., 2011). Additionally, a number of major malformations can be also induced by SMG9 dysfunction (Shaheen et al., 2016). Shaheed's study suggested SMG9 played an important role in NMD process (Shaheen et al., 2016), However, insufficient researches about SMG9 has so far hampered a molecular understanding of the NMD mechanisms. Recent studies reported that microRNAs are involved in NMD pathway by targeting several NMD factors. For example, miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51 to promotes neural differentiation (Bruno et al., 2011). Moreover, miR125a and miR125b could bind 3’UTR of SMG1 to regulate expression of SMG1, and consequently to regulate NMD activity (Wang et al., 2013a). Furthermore, miR-433 could regulate SMG5 expression by targeting to 3’UTR of SMG5 so that regulated NMD pathway (Jin et al., 2016). These studies suggest the existence of a conserved mRNA circuit that links the microRNA and NMD pathways resulting in the induction of cell type-specific transcripts during development. Here we demonstrated that miR-4651 represses the expression of SMG9 by degradation of the SMG9 mRNA. Subsequently, SMG9 downregulation inhibits the NMD activity. Therefore, this study supports a miR-4651/NMD regulatory circuit in eukaryotic cells.
The Hela cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM; Hyclone; Logan; Utah; USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; Logan; Utah; USA) at 37 °C in a humidified atmosphere of 5% CO2. 2.3. Transfection Hela cells were incubated in 5% CO2 at 37 °C in DMEM (Hyclone; Logan; Utah; USA) supplemented with 10% FBS (Hyclone; Logan; Utah; USA). One day before transfection, 1 × 105 cells were plated in 2 mL DMEM containing 10% FBS per well of a 6-well plate. The next day, when the cells confluence get 50%, the medium was changed to OptiMEM I Reduced Serum Medium (Hyclone; Logan; Utah; USA), and cells were transfected with overexpression vector (SMG9, pmirGLO-SMG9WT, pmirGLO-SMG9-MUT, 4 μg) or siRNA (siSMG9, siNC, 10 μL) or miRNA (miR-4651 mimic, inhibitor, 10 μL) or NMD reporter plasmid (4 μg) using 10 μL Lipo6000™ reagent (#C0526; Beyotime; Nanjing; China). After 4–6 h, medium was replaced by DMEM containing 10% FBS (Hyclone; Logan; Utah; USA). The cells were incubated for 24–48 h. Transfected cells were washed twice with cold PBS (Hyclone; Logan; Utah; USA). At least three independent experiments were performed for each assay. Opti-MEM I Reduced Serum Medium (Hyclone; Logan; Utah; USA) was used as diluent. 2.4. Dual-luciferase reporter assays The luciferase reporter gene vector, which includes the SMG9 sequences containing miR-4651 binding sites, was designed according to the SMG9 sequences and the predicted result of the Target Scan software (http://www.tar-getscan.org/). The wild type (CCACCCC) or a mutant seed sequence (CAGTACC) of SMG9 was inserted into the pmirGLO luciferase reporter vector to construct pmirGLO-SMG9-WT and pmirGLO-SMG9-mut vectors. Luciferase enzymatic activities were measured with PerkinElmer 2030 Multilabel Reader (PerkinElmer; German) using the dual-luciferase reporter assay system (Promega; Beijing; China). The transfection experiment was performed in triplicates and the data were expressed as means ± SD.
2. Materials and methods 2.1. Plasmids construction and synthesis of miRNA and siRNA
2.5. RNA extraction, RT-PCR and real-time quantitative RT-PCR The SMG9 overexpression vector was constructed by using seamless cloning kit (#C112-01; ClonExpress II One Step Cloning Kit; Vazyme; Nanjing; China). Briefly, the primers used to amplify the region of SMG9 were designed by CE Design V1.04 (Vazyme; Nanjing; China), and the sequence was F: 5′-ctagcgtttaaacttaagcttATGTCTGAGTCTGGA CACAGTCAGC-3′ and R: 5′-ccacactggactagtggatccTCAGGCCAGCAGGC GGCT-3′. Then the complete region of SMG9 was amplified by PCR with KOD-Plus-Neo (TOYOBO; Japan). The production of PCR was validated by sequencing (AUGCT; Wuhan; China). Then the PCR production was cloned into the digested pcDNA3.1 vector (HindIII and BamHI enzyme) according to the constructions. The overexpression vector was validated by sequencing. NMD activity was detected with an NMD reporter plasmid, which was constructed according to methods described by Pereverzev AP et al. (Pereverzev et al., 2015). pcDNA3.1 was used to induce another promoter system (SV40) driving enhanced green fluorescent protein (EGFP) expression. Then, mutant β-globin, which can be degraded by NMD, was inserted between the SV40 promoter and EGFP. mCherry expression was driven by the CMV promoter. Therefore, the activity of NMD was shown by the expression ratio of EGFP and mCherry. The miRNA and siRNA were designed and synthesized by genepharma (Shanghai; China). The completeness and quality of both siRNA and miRNA were validated by electrophoresis. The miRNA sequence was listed in Table S1.
Total RNA was extracted with TRizol ® Reagent (Invitrogen; Carlsbad; CA; USA) according to manufacturer's instructions. The synthesis of the cDNA was carried out with Primescript RT Reagent Kit with gDNA Eraser (#FSQ-201; TOYOBO; Japan). The primers sequences used in the qRT-PCR were listed in Table S2. Real time quantitative PCR was performed with SYBR Green I Real-time PCR Master Mix (TOYOBO; Japan) on a LightCycler® 480 Real-time System (Roche). The expression of target genes was normalized by that of β-actin; Relative gene expression was calculated using 2-ΔΔCt method. 2.6. Protein extractions and Western blotting analysis The whole-cell protein of Hela was collected using RIPA and PMSF, and conserved at −80 °C. The concentration of protein was detected using the bicinchoninic acid (BCA) method (Beyotime Biotechnology; Jiangsu; China). Subsequently, samples were separated by SDS–PAGE (10%) and electroblotted onto polyvinylidene fluoride (PVDF) membrane (Millipore; USA). Membrane was blocked with 1× TBST supplemented with 5% skimmed milk powder (servicebio; Wuhan; China). Membranes were incubated with the primary antibody, anti-SMG9 (C17) (goat. #SC242114; Santa Cruz Biotechnology; Inc.; Delaware; USA). For normalization of the results, membranes were reblotted for GAPDH (goat; GB11002; servicebio; Wuhan; China). Proteins were 66
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4651 targeting SMG9. The wild report gene vector pmirGLO-SMG9-WT and miR-4651 mimic were co-transfected into Hela cells. Then we detected the normalized luciferase values, which showed 40% reduction compared with controls (P < 0.01, Fig. 1b). However, this regulation was abrogated when a four-nucleotide mutation (CACC–AGTA) was introduced in the miR-4651 seed sequence in SMG9 (Fig. 1b). Additionally, the luciferase activity in SMG9 mutant was increased compared to SMG9 WT (Fig. 1d). Furthermore, the dual-luciferase vectors and miR-4651 inhibitor were co-transfected into Hela cells. The result showed SMG9 expression was increased (Fig. 1c). This regulation was also abrogated when the pmirGLOSMG9-MUT was transfected (Fig. 1c). The results together showed that miR-4651 targets SMG9 directly.
detected with secondary antibody (HRP-rabbit; anti-goat; 1:1000; boster; Wuhan; China). For the TBL2 and GADD45B western blotting analysis, the primary antibodies were anti-TBL2 (L-15) (goat; #SC104692; Santa Cruz Biotechnology; Inc.; Delaware; USA), and antiGADD45B (rabbit; #ab128920; abcam; Inc.; Shanghai; China). Bands on the X-ray flms were quantifed with WCIF ImageJ software for the densitometry analysis. 2.7. Statistical analysis All the experiments were repeated for at least three times. All results were presented as means ± SD. Statistical significance was assessed using Student's t-test. P value < 0.05 was deemed to indicate statistical significance.
3.2. miR-4651 repressed SMG9 expression After we transfected miR-4651 mimic into Hela cells, the mRNA and protein expression levels of SMG9 were decreased (P < 0.01, Fig. 2a and b). Furthermore, the expression of SMG9 was increased significantly at both mRNA and protein level when the miR-4651 inhibitor was transfected into the Hela cells (P < 0.01, Fig. 2c and d). As the consequence, the SMG9 expression was down-regulated by miR-4651.
3. Results 3.1. miRNA-4651 targets of SMG9 We selected the possible miRNAs targeting SMG9 through miRNA algorithm DIANA TOOLS (http://diana.imis.athena-innovation.gr/ DianaTools/index.php). The prediction indicates that miR-4651 targets SMG9 (Fig. 1a). The CDS of SMG9 holds a sequence motif (CCAC CCC) complementary to the seed sequence GGUGGGG of miR-4651 (Fig. 1a). We performed the dual-luciferase assay to investigate miR-
3.3. The suppression of SMG9 repressed NMD activity Previous studies have indicated that SMG9 was an important NMD
Fig. 1. miR-4651 targets SMG9. a The predicated seed sequence of SMG9 by DIANA TOOLS (http://diana.imis.athena-innovation.gr/DianaTools/index.php). b Luciferase activity in Hela cells transfected with miR-4651 mimic or a NC miR-4651 mimic for 24 h. c Luciferase activity in Hela cells transfected with miR-4651 inhibitor or a NC inhibitor for 48 h. d Luciferase activity of the SMG9 WT vs SMG9 mutant in Hela cells transfected for 24 h. *P < 0.05, and ** P < 0.01. 67
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Fig. 2. miR-4651 reduced SMG9 protein and mRNA level. a, c qPCR analysis of Hela cells transfected with the miR-4651 mimic or inhibitor for 48 h. Shown are the results from three independent experiments, normalized to β-actin mRNA levels. **P < 0.01. b, d Western Blot analysis of endogenous SMG9 protein levels in Hela cells at 48 h after transfection with mature miR-4651 mimic or miR-4651 inhibitor. GAPDH was used as the internal control.
EGFP and mCherry of NMD reporter system, we found that miR-4651 mimic decreased NMD activity (Fig. 4c). Whereas, TBL2 and GADD45B were decreased in Hela cells when transfected with miR-4651 inhibitor (P < 0.05, Fig. 4d and e). Upon analysing the fluorescence intensity of EGFP and mCherry, we found that miR-4651 inhibitor enhanced NMD activity compared to control (Fig. 4f).
factor, therefore we suppressed SMG9 expression by RNAi to confrm this function. The siRNA was transfected into Hela cells for 24–48 h. The results showed that both mRNA and protein levels of SMG9 were decreased (Fig. 3a, c). According to the NMD mechanism, if NMD activity reduced, the mRNA levels of NMD substrates would be increased. The NMD activity thereby was indicated by the expression level of NMD substrates, TBL2 and GADD45B (Wang et al., 2013a; Jin et al., 2016). We detected the expression level of TBL2 and GADD45B, and then found that both of them were increased after SMG9 suppression (P < 0.05, Fig. 3b, c). To better illustrate the NMD efficiency, we introduced a NMD-reporter system, which was constructed according to methods described by Pereverzev AP et al. (Pereverzev et al., 2015). Upon analysing the fluorescence intensity of EGFP and mCherry, we found that NMD activity was decreased after SMG9 knockdown (Fig. 3d). To further investigate the effect of the SMG9 on NMD activity, the SMG9 was overexpressed. The overexpression efficiency was detected by the qRT-PCR method (Fig. 3e). Moreover, the expression level of TBL2 and GADD45B was detected to reflect the activity of NMD (Fig. 3f). To validate the expression level of these two NMD substrates, their protein level was also detected by Western Blot method, which showed that these two NMD substrates expression level was decreased by the overexpression of SMG9 (Fig. 3g). Then the NMD-reporter system was also used to detected the effect of SMG9 overexpression on NMD efficiency. The results showed that SMG9 overexpression decreased the ratio of the mRNA level of EGFP/mCherry significantly (p < 0.05). Consequently, the NMD activity was enhanced by the SMG9 overexpression.
4. Discussion NMD as an important mRNA quality surveillance system has been extensively studied recent years (Sheth and Parker, 2006; Yepiskoposyan et al., 2011). UPF1 is the key factor in NMD process, which play an important role in RNA binding and PTC recognition (Hilleren and Parker, 1999). UPF1 phosphorylation requires SMG1 catalysis, whereas SMG5, SMG6 and SMG7 were involved in UPF1 dephosphorylation (Pulak and Anderson, 1993; Page et al., 1999; Chiu et al., 2003; Ohnishi et al., 2003; Shukla et al., 2011). Both SMG8 and SMG9 bind to SMG1 to participate in UPF1 phosphorylation (Yamashita et al., 2009). Our results showed that NMD activity was repressed when the expression of SMG9 was suppressed. Therefore, the regulation to SMG9 could affect NMD activity. Many studies have shown that miRNAs can inhibit the translation of their target mRNAs or promote its degradation (Shukla et al., 2011). Recent analysis of depletion of protein production indicates that mammalian miRNAs primarily reduce target mRNA levels through miRNA-mediated target mRNA destabilization (Guo et al., 2010). We found a microRNA, miR-4651, targeting to SMG9 by bioinformatic prediction. The regulation of miR-4651 targeting SMG9 was detected by dual-luciferase report assay. The result indicated that SMG9 expression was reduced by 40% when we transfected with miR-4651 mimic, whereas the miR-4651 inhibitor increased SMG9 mRNA levels. Our results suggested that miR-4651 decreased SMG9 levels by degradation of the SMG9 mRNA. It has been demonstrated that the NMD activity was repressed when suppression of SMG9 (Fig. 3). Therefore, miRNA4651 repressed NMD activity by suppressing SMG9 expression.
3.4. miR-4651 repressed NMD activity We have demonstrated that miR-4651 repressed SMG9 expression so that to repress NMD activity. Indeed, our results showed that the NMD substrates TBL2 and GADD45B were increased in Hela cells at mRNA and protein level when transfected with miR-4651 mimic (P < 0.05, Fig. 4a and b). Upon analysing the fluorescence intensity of 68
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Fig. 3. Knockdown SMG9 expression repressed NMD activity. a qPCR analysis of SMG9 mRNA expression in Hela cells transfected with RNA interference or NC fragments for 24 h. b qPCR analysis showed the two NMD substrates, TBL2 and GADD45B, mRNA level in Hela cells at 24 h after transfection with the RNA interference or NC fragment. c Western Blot analysis of endogenous SMG9 protein levels in Hela cells at 48 h after transfection with the RNA interference or NC fragment. GAPDH was used as the internal control. d NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was decreased after SMG9 was inhibited. e qPCR analysis of SMG9 mRNA expression in Hela cells after SMG9 was overexpressed for 24 h. f qPCR analysis showed the two NMD substrates, TBL2 and GADD45B, mRNA level in Hela cells after SMG9 was overexpressed for 24 h. g Western Blot analysis of endogenous SMG9 protein levels in Hela cells after SMG9 was overexpressed for 48 h. GAPDH was used as the internal control. h NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was increased after SMG9 was overexpressed. All PCR data were normalized to β-actin mRNA expression and represent the average of three independent experiments ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.
2013b). These miRNA/NMD regulatory circuit were involved in many nervous system disorder diseases such as autism (Abuelneel et al., 2008), prion-induced neuron degeneration (Saba et al., 2008), Huntington's disease (Lee et al., 2011), Parkinson's disease (Kim et al., 2010), as well as Alzheimer's disease (Lukiw, 2007). These evidences indicate the presence of a microRNA/NMD regulatory circuit is involved in many human diseases and related therapies. We reported that miRNA-4651 repressed the expression of SMG9 by degradation of the SMG9 mRNA. This down-regulation of SMG9 by miRNA-4651 also inhibited the NMD pathway. These results suggest that there exists a miR-4651/NMD regulatory circuit, which may help the management of diseases diagnosis and related treatment.
NMD has been found to be a highly regulated pathway. There is increasing evidence that NMD plays a vital role in many biological processes. Loss of NMD leads to developmental defects (Huang and Wilkinson, 2012). Studies on mammalian cell lines have shown that NMD is essential for specific steps in neuro development (Lou et al., 2014). In human, mutation of UPF3B, the NMD factor, causes intellectual disability (Nguyen et al., 2014). These cognitive impairments may be caused by developmental defects. Furthermore, loss of the NMD factor UPF2 disrupts hematopoiesis and liver development in vivo (Weischenfeldt et al., 2008; Thoren et al., 2010), and moreover, there is evidence that NMD cooperates with another RNA decay pathway to affect myoblast differentiation (Gong et al., 2009). It has recently been reported that miR-128 inhibits NMD function by targeting UPF1 and EJC core component MLN51 in mammals (Bruno et al., 2011). And additionally, miRNA-125 (miRNA-125a and miRNA-125b) inhibits SMG1 expression and thereby inhibits the NMD pathway (Wang et al.,
5. Conclusion Our study indicated that NMD activity is inhibited by miR-4651. 69
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Fig. 4. miR-4651 repressed NMD activity. a qPCR analysis of two NMD substrates, TBL2 and GADD45B relative mRNA expression level in Hela cells at 24 h after transfection with the miR-4651 mimic. b Western Blot analysis of endogenous NMD substrates, TBL2 and GADD45B protein levels in cells at 48 h after transfection with the miR-4651 mimic. c NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was decreased after transfection with the miR-4651 mimic for 48 h. d qPCR analysis of two NMD substrates, TBL2 and GADD45B relative mRNA expression level in Hela cells at 24 h after transfection with the miR-4651 inhibitor. e Western Blot analysis of endogenous NMD substrates, TBL2 and GADD45B protein levels in Hela cells at 48 h after transfection with the miR-4651 inhibitor. f NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was increased after transfection with the miR-4651 inhibitor for 48 h. All PCR data were normalized to β-actin mRNA expression and represent the average of three independent experiments ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.
Mechanistically, miR-4651 targets SMG9 and inhibits the expression of SMG9, so that to suppress NMD activity. This funding provides evidence for microRNA/NMD regulatory mechanism and therapeutic advice for NMD-related diseases.
YJT contributed reagents/materials/analysis tools. YJT wrote the manuscript.
Funding Competing interests This work was supported by the Fundamental Research Funds for the Central Universities (No. 2662018PY043), the Applied Basic Research Programs of Science and Technology of Wuhan, China (No. 2016020101010091), the National Key Technology Support Program of China (No. 2015BAI09B06) and the National Project for Breeding of Transgenic Pig (No. 2016ZX08006-002).
The authors have declared that no competing interests exist. Data availability All data are fully available without restriction. All relevant data are within the paper.
Ethics approval and consent to participate
Author's contributions
Not applicable.
ZFM & ZQR conceived and designed the experiments. ZFM performed experiments. ZFM, YJ and YJT analyzed data. ZFM, YJ, RJZ and 70
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Consent to publish
Lukiw, W.J., 2007. Micro-RNA speciation in fetal, adult and Alzheimer's disease hippocampus. Neuroreport 18, 297. Lykkeandersen, J., Bennett, E.J., 2014. Protecting the proteome: eukaryotic cotranslational quality control pathways. J. Cell Biol. 204, 467–476. Melero, R., Uchiyama, A., Castaño, R., Kataoka, N., Kurosawa, H., Ohno, S., Yamashita, A., Llorca, O., 2014. Structures of SMG1-UPFs complexes: SMG1 contributes to regulate UPF2-dependent activation of UPF1 in NMD. Structure 22, 1105–1119. Nguyen, L.S., Wilkinson, M.F., Gecz, J., 2014. Nonsense-mediated mRNA decay: interindividual variability and human disease. Neurosci. Biobehav. Rev. 46, 175–186. Ohnishi, T., Yamashita, A., Kashima, I., Schell, T., Anders, K.R., Grimson, A., Hachiya, T., Hentze, M.W., Anderson, P., Ohno, S., 2003. Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7. Mol. Cell 12, 1187. Page, M.F., Carr, B., Anders, K.R., Grimson, A., Anderson, P., 1999. SMG-2 is a phosphorylated protein required for mRNA surveillance in Caenorhabditis elegans and related to Upf1p of yeast. Molecular & Cellular Biology 19, 5943–5951. Peccarelli, M., Kebaara, B.W., 2014. Regulation of natural mRNAs by the nonsensemediated mRNA decay pathway. Eukaryot. Cell 13, 1126–1135. Pereverzev, A.P., Gurskaya, N.G., Ermakova, G.V., Kudryavtseva, E.I., Markina, N.M., Kotlobay, A.A., Lukyanov, S.A., Zaraisky, A.G., Lukyanov, K.A., 2015. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci. Rep. 5, 7729. Popp, W.L., Maquat, L.E., 2013. Organizing principles of mammalian nonsense-mediated mRNA decay. Annu. Rev. Genet. 47, 139–165. Pulak, R., Anderson, P., 1993. mRNA surveillance by the Caenorhlabditis elegans smg genes. Genes Dev. 7, 1885–1897. Saba, R., Goodman, C.D., Huzarewich, R.L., Robertson, C., Booth, S.A., 2008. A miRNA signature of prion induced neurodegeneration. PLoS One 3, e3652. Schmidt, S.A., Foley, P.L., Jeong, D.H., Rymarquis, L.A., Doyle, F., Tenenbaum, S.A., Belasco, J.G., Green, P.J., 2015. Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells. Nucleic Acids Res. 43, 309–323. Shaheen, R., Anazi, S., Ben-Omran, T., Seidahmed, M.Z., Caddle, L.B., Palmer, K., Ali, R., Alshidi, T., Hagos, S., Goodwin, L., 2016. Mutations in SMG9, encoding an essential component of nonsense-mediated decay machinery, cause a multiple congenital anomaly syndrome in humans and mice. Am. J. Hum. Genet. 98, 643–652. Sheth, U., Parker, R., 2006. Targeting of aberrant mRNAs to cytoplasmic processing bodies. Cell 125, 1095. Shukla, G.C., Singh, J., Barik, S., 2011. MicroRNAs: processing, maturation, target recognition and regulatory functions. Molecular & Cellular Pharmacology 3, 83. Thoren, L.A., Nørgaard, G.A., Weischenfeldt, J., Waage, J., Jakobsen, J.S., Damgaard, I., Bergström, F.C., 2010. UPF2 is a critical regulator of liver development. Function and Regeneration. Plos One 5, e11650. Wang, G., Jiang, B., Jia, C., Chai, B., Liang, A., 2013a. MicroRNA 125 represses nonsensemediated mRNA decay by regulating SMG1 expression. Biochem. Biophys. Res. Commun. 435, 16–20. Wang, G., Jiang, B., Jia, C., Chai, B., Liang, A., 2013b. MicroRNA 125 represses nonsensemediated mRNA decay by regulating SMG1 expression. Biochemical & Biophysical Research Communications 435, 16–20. Weischenfeldt, J., Damgaard, I., Bryder, D., Theilgaardmönch, K., Thoren, L.A., Nielsen, F.C., Jacobsen, S.E., Nerlov, C., Porse, B.T., 2008. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements. Genes Dev. 22, 1381. Yamashita, A., Izumi, N., Kashima, I., Ohnishi, T., Saari, B., Katsuhata, Y., Muramatsu, R., Morita, T., Iwamatsu, A., Hachiya, T., 2009. SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay. Genes Dev. 23, 1091. Yepiskoposyan, H., Aeschimann, F., Nilsson, D., Okoniewski, M., Mühlemann, O., 2011. Autoregulation of the nonsense-mediated mRNA decay pathway in human cells. Rnaa publication of the Rna. Society 17, 2108.
Not applicable. Acknowledgements Not applicable. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.03.031. References Abuelneel, K., Liu, T., Gazzaniga, F.S., Nishimura, Y., Wall, D.P., Geschwind, D.H., Lao, K., Kosik, K.S., 2008. Heterogeneous dysregulation of microRNAs across the autism spectrum. Neurogenetics 9, 153–161. Bruno, I.G., Karam, R., Huang, L., Bhardwaj, A., Lou, C.H., Shum, E.Y., Song, H.W., Corbett, M.A., Gifford, W.D., Gecz, J., 2011. Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay. Mol. Cell 42, 500–510. Chang, Y.F., Imam, J.S., Wilkinson, M.F., 2007. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51–74. Chiu, S.Y., Serin, G., Ohara, O., Maquat, L.E., 2003. Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1. Rna-a publication of the Rna. Society 9, 77. Feng, Q., Jagannathan, S., Bradley, R.K., 2017. The RNA surveillance factor UPF1 represses myogenesis via its E3 ubiquitin ligase activity. Mol. Cell 67, 239. Fernà ndez, I.S., Yamashita, A., Arias-Palomo, E., Bamba, Y., Bartolomã©, R.A., Canales, M.A., Teixidã3, J., Ohno, S., Llorca, O., 2011. Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex. Nucleic Acids Res. 39, 347–358. Gong, C., Kim, Y.K., Woeller, C.F., Tang, Y., Maquat, L.E., 2009. SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs. Genes Dev. 23, 54. Guo, H., Ingolia, N.T., Weissman, J.S., Bartel, D.P., 2010. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840. Hilleren, P., Parker, R., 1999. mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization? RNA 5, 711–719. Huang, L., Wilkinson, M.F., 2012. Regulation of nonsense-mediated mRNA decay. Wiley Interdisciplinary Reviews Rna 3, 807–828. Jin, Y., Zhang, F., Ma, Z., Ren, Z., 2016. MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA. BMC Mol. Biol. 17, 17. Karousis, E.D., Nasif, S., Mühlemann, O., 2016. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact. Wiley Interdisciplinary Reviews Rna 7, 661. Kim, J., Inoue, K., Ishii, J., Vanti, W.B., Voronov, S.V., Murchison, E., Hannon, G., Abeliovich, A., 2010. A MicroRNA feedback circuit in midbrain dopamine neurons. Science 317, 1220–1224. Lee, S.T., Chu, K., Im, W.S., Yoon, H.J., Im, J.Y., Park, J.E., Park, K.H., Jung, K.H., Lee, S.K., Kim, M., 2011. Altered microRNA regulation in Huntington's disease models. Exp. Neurol. 227, 172–179. Lou, C.H., Shao, A., Shum, E.Y., Espinoza, J.L., Huang, L., Karam, R., Wilkinson, M.F., 2014. Posttranscriptional control of the stem cell and neurogenic programs by the nonsense-mediated RNA decay pathway. Cell Rep. 6, 748.
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Research paper
MicroRNA 4651 regulates nonsense-mediated mRNA decay by targeting SMG9 mRNA ⁎
Yanjie Tana, Zhenfa Maa, Yi Jina, Ruojun Zonga, Jian Wuc, , Zhuqing Rena,b,
T
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a Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China b The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China c College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-4651 SMG9 NMD
Nonsense-mediated mRNA decay (NMD) is originally identified as a conserved RNA surveillance mechanism that rapidly degrades aberrant mRNA containing premature termination codons (PTCs). However, the molecular regulation mechanisms by which microRNAs inhibit NMD has not been well understood. Here we identified that miR-4651 participated in the NMD pathway by downregulating expression levels of SMG9. We provided evidences that (1) Overexpression of miR-4651 mimic significantly inhibited the expression of SMG9 (P < 0.05); (2) NMD substrates genes, TBL2 and GADD45B were both increased at mRNA and protein expression levels when SMG9 was suppressed by siRNA, whereas decreased by SMG9 overexpression; (3) Expression of SMG9 was significantly increased but TBL2, GADD45B were significantly decreased when cells transfected with miR-4651 inhibitor (P < 0.05). These results indicated that miR-4651 regulated NMD by targeting SMG9 mRNA. Our study highlights that miR-4651 represses NMD. miR-4651 targets SMG9 and represses the expression levels of SMG9. NMD activity is decreased additionally when SMG9 is inhibited. The present study provides evidence for microRNA/NMD regulatory mechanism.
1. Introduction Eukaryotic gene expression involves a complex series of biological processes. From transcription of genetic information to protein synthesis, post-transcriptional modification and remodeling are essential for the translation of ribosomes into mature mRNAs (Fernà ndez et al., 2011). Several monitoring mechanisms ensure the fidelity and accuracy of these processes. Nonsense mediated mRNA decay (NMD) is a conservative surveillance mechanism of transcripts quality that recognizes and degrades aberrant transcripts containing a premature termination codons (PTC) (Chang et al., 2007; Popp and Maquat, 2013; Lykkeandersen and Bennett, 2014; Karousis et al., 2016). A quarter of PTCs are produced by mutations in human genetic diseases, e.g. cancers often exhibit abnormal transcription and mRNA processing. Therefore, NMD can protect cells from the accumulation of potentially harmful Cterminal truncated proteins encoded by PTC-containing mRNAs (Yamashita et al., 2009). In addition to DNA mutations, an error in transcription, or inefficiently spliced pre-mRNAs can also lead to
abnormal transcripts with PTC (Feng et al., 2017). NMD was also found to be responsible for degradation of 3–10% of normal transcripts in human, therefore serving as an widespread gene regulatory mechanism (Peccarelli and Kebaara, 2014). The machinery of NMD consists of the factors, UPF1, UPF2, UPF3, SMG1, SMG8 and SMG9 involved in PTC recognition, and SMG5, SMG6, SMG7 participating in the degradation of the target mRNAs (Schmidt et al., 2015). The presence of “surveillance complex”, discriminates PTC-containing mRNAs from normal mRNAs and triggers degradation of abnormal transcripts. A “surveillance complex” termed “SURF” was first assembled before the termination of translation. SURF includes SMG1, UPF1, eRF1, and eRF3 (Schmidt et al., 2015). If the exon junction complex (EJC) is located > 50–55 nucleotides downstream of the stop codon, the SURF and EJC interactions ensure that the termination code is identified as a PTC (Schmidt et al., 2015). This “judgment” followed by SMG1 forms the SMG1 complex (SMG1C) with SMG8 and SMG9 to phosphorylate UPF1. It is the single essential event to trigger all the later processes leading to the degradation of an mRNA (Melero
Abbreviations: SMG9, nonsense mediated mRNA decay associated PI3K related kinase9; NMD, nonsense mediated mRNA decay; UPF1, Up-framshift 1; PTC, premature termination codon; SMG1C, SMG1 complex; IDR, intrinsically disordered region; EJC, exon junction complex ⁎ Corresponding authors at: College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. E-mail addresses: [email protected] (J. Wu), [email protected] (Z. Ren). https://doi.org/10.1016/j.gene.2019.03.031 Received 24 December 2018; Received in revised form 11 March 2019; Accepted 17 March 2019 Available online 19 March 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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2.2. Cell culture
et al., 2014). SMG9 can inhibit SMG1 kinase activity in the isolated SMG1C, indicating that they regulate SMG1 during remodeling of the surveillance complex (Fernà ndez et al., 2011). SMG9 is a 520-amino acid protein that contains a nucleotide triphosphatase domain in the center. SMG9 could regulate the generation of SMG1C. Its N-terminal is a 180-residue internal unordered region (IDR) followed by a well-folded C-terminal domain, both of domains have the ability binding to SMG1 and stabilizing the integrity of the SMG1C (Fernà ndez et al., 2011). In humans and nematodes, SMG9 protein affects the stability of PTC-containing mRNA (Yamashita et al., 2009). Cells cannot recognize transcripts containing PTC with loss-offunction mutations of SMG9 (Shaheen et al., 2016). Patients with SMG9 deficiency exhibit extensive transcriptional disorders, suggesting that SMG9 plays an important role in post-transcriptional regulation and surveillance (Fernà ndez et al., 2011). Additionally, a number of major malformations can be also induced by SMG9 dysfunction (Shaheen et al., 2016). Shaheed's study suggested SMG9 played an important role in NMD process (Shaheen et al., 2016), However, insufficient researches about SMG9 has so far hampered a molecular understanding of the NMD mechanisms. Recent studies reported that microRNAs are involved in NMD pathway by targeting several NMD factors. For example, miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51 to promotes neural differentiation (Bruno et al., 2011). Moreover, miR125a and miR125b could bind 3’UTR of SMG1 to regulate expression of SMG1, and consequently to regulate NMD activity (Wang et al., 2013a). Furthermore, miR-433 could regulate SMG5 expression by targeting to 3’UTR of SMG5 so that regulated NMD pathway (Jin et al., 2016). These studies suggest the existence of a conserved mRNA circuit that links the microRNA and NMD pathways resulting in the induction of cell type-specific transcripts during development. Here we demonstrated that miR-4651 represses the expression of SMG9 by degradation of the SMG9 mRNA. Subsequently, SMG9 downregulation inhibits the NMD activity. Therefore, this study supports a miR-4651/NMD regulatory circuit in eukaryotic cells.
The Hela cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM; Hyclone; Logan; Utah; USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; Logan; Utah; USA) at 37 °C in a humidified atmosphere of 5% CO2. 2.3. Transfection Hela cells were incubated in 5% CO2 at 37 °C in DMEM (Hyclone; Logan; Utah; USA) supplemented with 10% FBS (Hyclone; Logan; Utah; USA). One day before transfection, 1 × 105 cells were plated in 2 mL DMEM containing 10% FBS per well of a 6-well plate. The next day, when the cells confluence get 50%, the medium was changed to OptiMEM I Reduced Serum Medium (Hyclone; Logan; Utah; USA), and cells were transfected with overexpression vector (SMG9, pmirGLO-SMG9WT, pmirGLO-SMG9-MUT, 4 μg) or siRNA (siSMG9, siNC, 10 μL) or miRNA (miR-4651 mimic, inhibitor, 10 μL) or NMD reporter plasmid (4 μg) using 10 μL Lipo6000™ reagent (#C0526; Beyotime; Nanjing; China). After 4–6 h, medium was replaced by DMEM containing 10% FBS (Hyclone; Logan; Utah; USA). The cells were incubated for 24–48 h. Transfected cells were washed twice with cold PBS (Hyclone; Logan; Utah; USA). At least three independent experiments were performed for each assay. Opti-MEM I Reduced Serum Medium (Hyclone; Logan; Utah; USA) was used as diluent. 2.4. Dual-luciferase reporter assays The luciferase reporter gene vector, which includes the SMG9 sequences containing miR-4651 binding sites, was designed according to the SMG9 sequences and the predicted result of the Target Scan software (http://www.tar-getscan.org/). The wild type (CCACCCC) or a mutant seed sequence (CAGTACC) of SMG9 was inserted into the pmirGLO luciferase reporter vector to construct pmirGLO-SMG9-WT and pmirGLO-SMG9-mut vectors. Luciferase enzymatic activities were measured with PerkinElmer 2030 Multilabel Reader (PerkinElmer; German) using the dual-luciferase reporter assay system (Promega; Beijing; China). The transfection experiment was performed in triplicates and the data were expressed as means ± SD.
2. Materials and methods 2.1. Plasmids construction and synthesis of miRNA and siRNA
2.5. RNA extraction, RT-PCR and real-time quantitative RT-PCR The SMG9 overexpression vector was constructed by using seamless cloning kit (#C112-01; ClonExpress II One Step Cloning Kit; Vazyme; Nanjing; China). Briefly, the primers used to amplify the region of SMG9 were designed by CE Design V1.04 (Vazyme; Nanjing; China), and the sequence was F: 5′-ctagcgtttaaacttaagcttATGTCTGAGTCTGGA CACAGTCAGC-3′ and R: 5′-ccacactggactagtggatccTCAGGCCAGCAGGC GGCT-3′. Then the complete region of SMG9 was amplified by PCR with KOD-Plus-Neo (TOYOBO; Japan). The production of PCR was validated by sequencing (AUGCT; Wuhan; China). Then the PCR production was cloned into the digested pcDNA3.1 vector (HindIII and BamHI enzyme) according to the constructions. The overexpression vector was validated by sequencing. NMD activity was detected with an NMD reporter plasmid, which was constructed according to methods described by Pereverzev AP et al. (Pereverzev et al., 2015). pcDNA3.1 was used to induce another promoter system (SV40) driving enhanced green fluorescent protein (EGFP) expression. Then, mutant β-globin, which can be degraded by NMD, was inserted between the SV40 promoter and EGFP. mCherry expression was driven by the CMV promoter. Therefore, the activity of NMD was shown by the expression ratio of EGFP and mCherry. The miRNA and siRNA were designed and synthesized by genepharma (Shanghai; China). The completeness and quality of both siRNA and miRNA were validated by electrophoresis. The miRNA sequence was listed in Table S1.
Total RNA was extracted with TRizol ® Reagent (Invitrogen; Carlsbad; CA; USA) according to manufacturer's instructions. The synthesis of the cDNA was carried out with Primescript RT Reagent Kit with gDNA Eraser (#FSQ-201; TOYOBO; Japan). The primers sequences used in the qRT-PCR were listed in Table S2. Real time quantitative PCR was performed with SYBR Green I Real-time PCR Master Mix (TOYOBO; Japan) on a LightCycler® 480 Real-time System (Roche). The expression of target genes was normalized by that of β-actin; Relative gene expression was calculated using 2-ΔΔCt method. 2.6. Protein extractions and Western blotting analysis The whole-cell protein of Hela was collected using RIPA and PMSF, and conserved at −80 °C. The concentration of protein was detected using the bicinchoninic acid (BCA) method (Beyotime Biotechnology; Jiangsu; China). Subsequently, samples were separated by SDS–PAGE (10%) and electroblotted onto polyvinylidene fluoride (PVDF) membrane (Millipore; USA). Membrane was blocked with 1× TBST supplemented with 5% skimmed milk powder (servicebio; Wuhan; China). Membranes were incubated with the primary antibody, anti-SMG9 (C17) (goat. #SC242114; Santa Cruz Biotechnology; Inc.; Delaware; USA). For normalization of the results, membranes were reblotted for GAPDH (goat; GB11002; servicebio; Wuhan; China). Proteins were 66
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4651 targeting SMG9. The wild report gene vector pmirGLO-SMG9-WT and miR-4651 mimic were co-transfected into Hela cells. Then we detected the normalized luciferase values, which showed 40% reduction compared with controls (P < 0.01, Fig. 1b). However, this regulation was abrogated when a four-nucleotide mutation (CACC–AGTA) was introduced in the miR-4651 seed sequence in SMG9 (Fig. 1b). Additionally, the luciferase activity in SMG9 mutant was increased compared to SMG9 WT (Fig. 1d). Furthermore, the dual-luciferase vectors and miR-4651 inhibitor were co-transfected into Hela cells. The result showed SMG9 expression was increased (Fig. 1c). This regulation was also abrogated when the pmirGLOSMG9-MUT was transfected (Fig. 1c). The results together showed that miR-4651 targets SMG9 directly.
detected with secondary antibody (HRP-rabbit; anti-goat; 1:1000; boster; Wuhan; China). For the TBL2 and GADD45B western blotting analysis, the primary antibodies were anti-TBL2 (L-15) (goat; #SC104692; Santa Cruz Biotechnology; Inc.; Delaware; USA), and antiGADD45B (rabbit; #ab128920; abcam; Inc.; Shanghai; China). Bands on the X-ray flms were quantifed with WCIF ImageJ software for the densitometry analysis. 2.7. Statistical analysis All the experiments were repeated for at least three times. All results were presented as means ± SD. Statistical significance was assessed using Student's t-test. P value < 0.05 was deemed to indicate statistical significance.
3.2. miR-4651 repressed SMG9 expression After we transfected miR-4651 mimic into Hela cells, the mRNA and protein expression levels of SMG9 were decreased (P < 0.01, Fig. 2a and b). Furthermore, the expression of SMG9 was increased significantly at both mRNA and protein level when the miR-4651 inhibitor was transfected into the Hela cells (P < 0.01, Fig. 2c and d). As the consequence, the SMG9 expression was down-regulated by miR-4651.
3. Results 3.1. miRNA-4651 targets of SMG9 We selected the possible miRNAs targeting SMG9 through miRNA algorithm DIANA TOOLS (http://diana.imis.athena-innovation.gr/ DianaTools/index.php). The prediction indicates that miR-4651 targets SMG9 (Fig. 1a). The CDS of SMG9 holds a sequence motif (CCAC CCC) complementary to the seed sequence GGUGGGG of miR-4651 (Fig. 1a). We performed the dual-luciferase assay to investigate miR-
3.3. The suppression of SMG9 repressed NMD activity Previous studies have indicated that SMG9 was an important NMD
Fig. 1. miR-4651 targets SMG9. a The predicated seed sequence of SMG9 by DIANA TOOLS (http://diana.imis.athena-innovation.gr/DianaTools/index.php). b Luciferase activity in Hela cells transfected with miR-4651 mimic or a NC miR-4651 mimic for 24 h. c Luciferase activity in Hela cells transfected with miR-4651 inhibitor or a NC inhibitor for 48 h. d Luciferase activity of the SMG9 WT vs SMG9 mutant in Hela cells transfected for 24 h. *P < 0.05, and ** P < 0.01. 67
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Fig. 2. miR-4651 reduced SMG9 protein and mRNA level. a, c qPCR analysis of Hela cells transfected with the miR-4651 mimic or inhibitor for 48 h. Shown are the results from three independent experiments, normalized to β-actin mRNA levels. **P < 0.01. b, d Western Blot analysis of endogenous SMG9 protein levels in Hela cells at 48 h after transfection with mature miR-4651 mimic or miR-4651 inhibitor. GAPDH was used as the internal control.
EGFP and mCherry of NMD reporter system, we found that miR-4651 mimic decreased NMD activity (Fig. 4c). Whereas, TBL2 and GADD45B were decreased in Hela cells when transfected with miR-4651 inhibitor (P < 0.05, Fig. 4d and e). Upon analysing the fluorescence intensity of EGFP and mCherry, we found that miR-4651 inhibitor enhanced NMD activity compared to control (Fig. 4f).
factor, therefore we suppressed SMG9 expression by RNAi to confrm this function. The siRNA was transfected into Hela cells for 24–48 h. The results showed that both mRNA and protein levels of SMG9 were decreased (Fig. 3a, c). According to the NMD mechanism, if NMD activity reduced, the mRNA levels of NMD substrates would be increased. The NMD activity thereby was indicated by the expression level of NMD substrates, TBL2 and GADD45B (Wang et al., 2013a; Jin et al., 2016). We detected the expression level of TBL2 and GADD45B, and then found that both of them were increased after SMG9 suppression (P < 0.05, Fig. 3b, c). To better illustrate the NMD efficiency, we introduced a NMD-reporter system, which was constructed according to methods described by Pereverzev AP et al. (Pereverzev et al., 2015). Upon analysing the fluorescence intensity of EGFP and mCherry, we found that NMD activity was decreased after SMG9 knockdown (Fig. 3d). To further investigate the effect of the SMG9 on NMD activity, the SMG9 was overexpressed. The overexpression efficiency was detected by the qRT-PCR method (Fig. 3e). Moreover, the expression level of TBL2 and GADD45B was detected to reflect the activity of NMD (Fig. 3f). To validate the expression level of these two NMD substrates, their protein level was also detected by Western Blot method, which showed that these two NMD substrates expression level was decreased by the overexpression of SMG9 (Fig. 3g). Then the NMD-reporter system was also used to detected the effect of SMG9 overexpression on NMD efficiency. The results showed that SMG9 overexpression decreased the ratio of the mRNA level of EGFP/mCherry significantly (p < 0.05). Consequently, the NMD activity was enhanced by the SMG9 overexpression.
4. Discussion NMD as an important mRNA quality surveillance system has been extensively studied recent years (Sheth and Parker, 2006; Yepiskoposyan et al., 2011). UPF1 is the key factor in NMD process, which play an important role in RNA binding and PTC recognition (Hilleren and Parker, 1999). UPF1 phosphorylation requires SMG1 catalysis, whereas SMG5, SMG6 and SMG7 were involved in UPF1 dephosphorylation (Pulak and Anderson, 1993; Page et al., 1999; Chiu et al., 2003; Ohnishi et al., 2003; Shukla et al., 2011). Both SMG8 and SMG9 bind to SMG1 to participate in UPF1 phosphorylation (Yamashita et al., 2009). Our results showed that NMD activity was repressed when the expression of SMG9 was suppressed. Therefore, the regulation to SMG9 could affect NMD activity. Many studies have shown that miRNAs can inhibit the translation of their target mRNAs or promote its degradation (Shukla et al., 2011). Recent analysis of depletion of protein production indicates that mammalian miRNAs primarily reduce target mRNA levels through miRNA-mediated target mRNA destabilization (Guo et al., 2010). We found a microRNA, miR-4651, targeting to SMG9 by bioinformatic prediction. The regulation of miR-4651 targeting SMG9 was detected by dual-luciferase report assay. The result indicated that SMG9 expression was reduced by 40% when we transfected with miR-4651 mimic, whereas the miR-4651 inhibitor increased SMG9 mRNA levels. Our results suggested that miR-4651 decreased SMG9 levels by degradation of the SMG9 mRNA. It has been demonstrated that the NMD activity was repressed when suppression of SMG9 (Fig. 3). Therefore, miRNA4651 repressed NMD activity by suppressing SMG9 expression.
3.4. miR-4651 repressed NMD activity We have demonstrated that miR-4651 repressed SMG9 expression so that to repress NMD activity. Indeed, our results showed that the NMD substrates TBL2 and GADD45B were increased in Hela cells at mRNA and protein level when transfected with miR-4651 mimic (P < 0.05, Fig. 4a and b). Upon analysing the fluorescence intensity of 68
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Fig. 3. Knockdown SMG9 expression repressed NMD activity. a qPCR analysis of SMG9 mRNA expression in Hela cells transfected with RNA interference or NC fragments for 24 h. b qPCR analysis showed the two NMD substrates, TBL2 and GADD45B, mRNA level in Hela cells at 24 h after transfection with the RNA interference or NC fragment. c Western Blot analysis of endogenous SMG9 protein levels in Hela cells at 48 h after transfection with the RNA interference or NC fragment. GAPDH was used as the internal control. d NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was decreased after SMG9 was inhibited. e qPCR analysis of SMG9 mRNA expression in Hela cells after SMG9 was overexpressed for 24 h. f qPCR analysis showed the two NMD substrates, TBL2 and GADD45B, mRNA level in Hela cells after SMG9 was overexpressed for 24 h. g Western Blot analysis of endogenous SMG9 protein levels in Hela cells after SMG9 was overexpressed for 48 h. GAPDH was used as the internal control. h NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was increased after SMG9 was overexpressed. All PCR data were normalized to β-actin mRNA expression and represent the average of three independent experiments ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.
2013b). These miRNA/NMD regulatory circuit were involved in many nervous system disorder diseases such as autism (Abuelneel et al., 2008), prion-induced neuron degeneration (Saba et al., 2008), Huntington's disease (Lee et al., 2011), Parkinson's disease (Kim et al., 2010), as well as Alzheimer's disease (Lukiw, 2007). These evidences indicate the presence of a microRNA/NMD regulatory circuit is involved in many human diseases and related therapies. We reported that miRNA-4651 repressed the expression of SMG9 by degradation of the SMG9 mRNA. This down-regulation of SMG9 by miRNA-4651 also inhibited the NMD pathway. These results suggest that there exists a miR-4651/NMD regulatory circuit, which may help the management of diseases diagnosis and related treatment.
NMD has been found to be a highly regulated pathway. There is increasing evidence that NMD plays a vital role in many biological processes. Loss of NMD leads to developmental defects (Huang and Wilkinson, 2012). Studies on mammalian cell lines have shown that NMD is essential for specific steps in neuro development (Lou et al., 2014). In human, mutation of UPF3B, the NMD factor, causes intellectual disability (Nguyen et al., 2014). These cognitive impairments may be caused by developmental defects. Furthermore, loss of the NMD factor UPF2 disrupts hematopoiesis and liver development in vivo (Weischenfeldt et al., 2008; Thoren et al., 2010), and moreover, there is evidence that NMD cooperates with another RNA decay pathway to affect myoblast differentiation (Gong et al., 2009). It has recently been reported that miR-128 inhibits NMD function by targeting UPF1 and EJC core component MLN51 in mammals (Bruno et al., 2011). And additionally, miRNA-125 (miRNA-125a and miRNA-125b) inhibits SMG1 expression and thereby inhibits the NMD pathway (Wang et al.,
5. Conclusion Our study indicated that NMD activity is inhibited by miR-4651. 69
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Fig. 4. miR-4651 repressed NMD activity. a qPCR analysis of two NMD substrates, TBL2 and GADD45B relative mRNA expression level in Hela cells at 24 h after transfection with the miR-4651 mimic. b Western Blot analysis of endogenous NMD substrates, TBL2 and GADD45B protein levels in cells at 48 h after transfection with the miR-4651 mimic. c NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was decreased after transfection with the miR-4651 mimic for 48 h. d qPCR analysis of two NMD substrates, TBL2 and GADD45B relative mRNA expression level in Hela cells at 24 h after transfection with the miR-4651 inhibitor. e Western Blot analysis of endogenous NMD substrates, TBL2 and GADD45B protein levels in Hela cells at 48 h after transfection with the miR-4651 inhibitor. f NMD reporter plasmid was used to detect the NMD activity, the result showed that the NMD activity was increased after transfection with the miR-4651 inhibitor for 48 h. All PCR data were normalized to β-actin mRNA expression and represent the average of three independent experiments ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.
Mechanistically, miR-4651 targets SMG9 and inhibits the expression of SMG9, so that to suppress NMD activity. This funding provides evidence for microRNA/NMD regulatory mechanism and therapeutic advice for NMD-related diseases.
YJT contributed reagents/materials/analysis tools. YJT wrote the manuscript.
Funding Competing interests This work was supported by the Fundamental Research Funds for the Central Universities (No. 2662018PY043), the Applied Basic Research Programs of Science and Technology of Wuhan, China (No. 2016020101010091), the National Key Technology Support Program of China (No. 2015BAI09B06) and the National Project for Breeding of Transgenic Pig (No. 2016ZX08006-002).
The authors have declared that no competing interests exist. Data availability All data are fully available without restriction. All relevant data are within the paper.
Ethics approval and consent to participate
Author's contributions
Not applicable.
ZFM & ZQR conceived and designed the experiments. ZFM performed experiments. ZFM, YJ and YJT analyzed data. ZFM, YJ, RJZ and 70
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Consent to publish
Lukiw, W.J., 2007. Micro-RNA speciation in fetal, adult and Alzheimer's disease hippocampus. Neuroreport 18, 297. Lykkeandersen, J., Bennett, E.J., 2014. Protecting the proteome: eukaryotic cotranslational quality control pathways. J. Cell Biol. 204, 467–476. Melero, R., Uchiyama, A., Castaño, R., Kataoka, N., Kurosawa, H., Ohno, S., Yamashita, A., Llorca, O., 2014. Structures of SMG1-UPFs complexes: SMG1 contributes to regulate UPF2-dependent activation of UPF1 in NMD. Structure 22, 1105–1119. Nguyen, L.S., Wilkinson, M.F., Gecz, J., 2014. Nonsense-mediated mRNA decay: interindividual variability and human disease. Neurosci. Biobehav. Rev. 46, 175–186. Ohnishi, T., Yamashita, A., Kashima, I., Schell, T., Anders, K.R., Grimson, A., Hachiya, T., Hentze, M.W., Anderson, P., Ohno, S., 2003. Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7. Mol. Cell 12, 1187. Page, M.F., Carr, B., Anders, K.R., Grimson, A., Anderson, P., 1999. SMG-2 is a phosphorylated protein required for mRNA surveillance in Caenorhabditis elegans and related to Upf1p of yeast. Molecular & Cellular Biology 19, 5943–5951. Peccarelli, M., Kebaara, B.W., 2014. Regulation of natural mRNAs by the nonsensemediated mRNA decay pathway. Eukaryot. Cell 13, 1126–1135. Pereverzev, A.P., Gurskaya, N.G., Ermakova, G.V., Kudryavtseva, E.I., Markina, N.M., Kotlobay, A.A., Lukyanov, S.A., Zaraisky, A.G., Lukyanov, K.A., 2015. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci. Rep. 5, 7729. Popp, W.L., Maquat, L.E., 2013. Organizing principles of mammalian nonsense-mediated mRNA decay. Annu. Rev. Genet. 47, 139–165. Pulak, R., Anderson, P., 1993. mRNA surveillance by the Caenorhlabditis elegans smg genes. Genes Dev. 7, 1885–1897. Saba, R., Goodman, C.D., Huzarewich, R.L., Robertson, C., Booth, S.A., 2008. A miRNA signature of prion induced neurodegeneration. PLoS One 3, e3652. Schmidt, S.A., Foley, P.L., Jeong, D.H., Rymarquis, L.A., Doyle, F., Tenenbaum, S.A., Belasco, J.G., Green, P.J., 2015. Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells. Nucleic Acids Res. 43, 309–323. Shaheen, R., Anazi, S., Ben-Omran, T., Seidahmed, M.Z., Caddle, L.B., Palmer, K., Ali, R., Alshidi, T., Hagos, S., Goodwin, L., 2016. Mutations in SMG9, encoding an essential component of nonsense-mediated decay machinery, cause a multiple congenital anomaly syndrome in humans and mice. Am. J. Hum. Genet. 98, 643–652. Sheth, U., Parker, R., 2006. Targeting of aberrant mRNAs to cytoplasmic processing bodies. Cell 125, 1095. Shukla, G.C., Singh, J., Barik, S., 2011. MicroRNAs: processing, maturation, target recognition and regulatory functions. Molecular & Cellular Pharmacology 3, 83. Thoren, L.A., Nørgaard, G.A., Weischenfeldt, J., Waage, J., Jakobsen, J.S., Damgaard, I., Bergström, F.C., 2010. UPF2 is a critical regulator of liver development. Function and Regeneration. Plos One 5, e11650. Wang, G., Jiang, B., Jia, C., Chai, B., Liang, A., 2013a. MicroRNA 125 represses nonsensemediated mRNA decay by regulating SMG1 expression. Biochem. Biophys. Res. Commun. 435, 16–20. Wang, G., Jiang, B., Jia, C., Chai, B., Liang, A., 2013b. MicroRNA 125 represses nonsensemediated mRNA decay by regulating SMG1 expression. Biochemical & Biophysical Research Communications 435, 16–20. Weischenfeldt, J., Damgaard, I., Bryder, D., Theilgaardmönch, K., Thoren, L.A., Nielsen, F.C., Jacobsen, S.E., Nerlov, C., Porse, B.T., 2008. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements. Genes Dev. 22, 1381. Yamashita, A., Izumi, N., Kashima, I., Ohnishi, T., Saari, B., Katsuhata, Y., Muramatsu, R., Morita, T., Iwamatsu, A., Hachiya, T., 2009. SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay. Genes Dev. 23, 1091. Yepiskoposyan, H., Aeschimann, F., Nilsson, D., Okoniewski, M., Mühlemann, O., 2011. Autoregulation of the nonsense-mediated mRNA decay pathway in human cells. Rnaa publication of the Rna. Society 17, 2108.
Not applicable. Acknowledgements Not applicable. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.03.031. References Abuelneel, K., Liu, T., Gazzaniga, F.S., Nishimura, Y., Wall, D.P., Geschwind, D.H., Lao, K., Kosik, K.S., 2008. Heterogeneous dysregulation of microRNAs across the autism spectrum. Neurogenetics 9, 153–161. Bruno, I.G., Karam, R., Huang, L., Bhardwaj, A., Lou, C.H., Shum, E.Y., Song, H.W., Corbett, M.A., Gifford, W.D., Gecz, J., 2011. Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay. Mol. Cell 42, 500–510. Chang, Y.F., Imam, J.S., Wilkinson, M.F., 2007. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51–74. Chiu, S.Y., Serin, G., Ohara, O., Maquat, L.E., 2003. Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1. Rna-a publication of the Rna. Society 9, 77. Feng, Q., Jagannathan, S., Bradley, R.K., 2017. The RNA surveillance factor UPF1 represses myogenesis via its E3 ubiquitin ligase activity. Mol. Cell 67, 239. Fernà ndez, I.S., Yamashita, A., Arias-Palomo, E., Bamba, Y., Bartolomã©, R.A., Canales, M.A., Teixidã3, J., Ohno, S., Llorca, O., 2011. Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex. Nucleic Acids Res. 39, 347–358. Gong, C., Kim, Y.K., Woeller, C.F., Tang, Y., Maquat, L.E., 2009. SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs. Genes Dev. 23, 54. Guo, H., Ingolia, N.T., Weissman, J.S., Bartel, D.P., 2010. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840. Hilleren, P., Parker, R., 1999. mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization? RNA 5, 711–719. Huang, L., Wilkinson, M.F., 2012. Regulation of nonsense-mediated mRNA decay. Wiley Interdisciplinary Reviews Rna 3, 807–828. Jin, Y., Zhang, F., Ma, Z., Ren, Z., 2016. MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA. BMC Mol. Biol. 17, 17. Karousis, E.D., Nasif, S., Mühlemann, O., 2016. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact. Wiley Interdisciplinary Reviews Rna 7, 661. Kim, J., Inoue, K., Ishii, J., Vanti, W.B., Voronov, S.V., Murchison, E., Hannon, G., Abeliovich, A., 2010. A MicroRNA feedback circuit in midbrain dopamine neurons. Science 317, 1220–1224. Lee, S.T., Chu, K., Im, W.S., Yoon, H.J., Im, J.Y., Park, J.E., Park, K.H., Jung, K.H., Lee, S.K., Kim, M., 2011. Altered microRNA regulation in Huntington's disease models. Exp. Neurol. 227, 172–179. Lou, C.H., Shao, A., Shum, E.Y., Espinoza, J.L., Huang, L., Karam, R., Wilkinson, M.F., 2014. Posttranscriptional control of the stem cell and neurogenic programs by the nonsense-mediated RNA decay pathway. Cell Rep. 6, 748.
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