Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Zhang, Xu; Lei, Yingfeng

doi:10.3390/ijms160922456

Cited by 19 publications

(11 citation statements)

References 46 publications

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“…RNA pulldown assay. A549 cell lines that ectopically expressed S1-lnc-ISG20, S1-lnc-ISG20-mut, or control cells were harvested for pulldown assays as previously described (40). Briefly, cells were UV cross-linked and lysed with lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride) with RNase inhibitor for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Chai

Shangguan

et al. 2018

J Virol

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Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication. The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

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Section: Methodsmentioning

confidence: 99%

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Chai

Shangguan

et al. 2018

J Virol

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“…Plasmids were constructed as follows: S1‐tagged lnc‐HUR1 plasmid cytomegalovirus (CMV)‐lnc‐HUR1‐S1 was constructed by cloning lnc‐HUR1 and S1 into pFLAG‐CMV2 vector; pCMV‐lnc‐HUR1 was constructed by cloning lnc‐HUR1 into pcDNA3.1 vector; and pGL2‐HUR1‐promoter was constructed by cloning predicted HUR1 promoter (genome: nt73324591‐73324941) to pGL2‐Basic vector. Streptavidin T1 magnetic beads were purchased from Invitrogen (USA).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus–Upregulated LNC‐HUR1 Promotes Cell Proliferation and Tumorigenesis by Blocking p53 Activity

Liu

Yang

et al. 2018

Hepatology

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Recent studies have indicated that a number of long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma, while their aberrant expressions are associated with tumorigenesis and poor prognosis. To identify hepatitis B virus (HBV)-related lncRNAs, we used RNA deep sequencing to quantify the abundances of lncRNAs in HepG2 cells and HBV transgenic HepG2-4D14 cells. Here, we demonstrate that lnc-HUR1 is significantly upregulated in HepG2-4D14 cells. We found that HBV-encoded hepatitis B x protein can enhance the transcription of lnc-HUR1. Overexpression of lnc-HUR1 promotes cell proliferation, whereas knockdown of lnc-HUR1 inhibits cell growth. We identified that lnc-HUR1 can interact with p53 and inhibit its transcriptional regulation on downstream genes, such as p21 and B cell lymphoma 2-associated X protein. We generated lnc-HUR1 transgenic mice and performed the partial hepatectomy (PHx) to examine liver regeneration. The data showed that the ratio of liver weight to body weight in lnc-HUR1 transgenic mice is higher than that in wild-type (WT) littermates at day 2 and day 3 following hepatectomy. Consistently, the results of bromodeoxyuridine staining on liver sections following hepatectomy indicate that the ratio of bromodeoxyuridine-positive cells in lnc-HUR1 transgenic mice is significantly higher than that in WT mice, suggesting that lnc-HUR1 promotes cell proliferation during liver regeneration. Next, we performed the experiment of diethylnitrosamine-induced tumorigenesis. The data demonstrate that tumor number in lnc-HUR1 transgenic mice is higher compared with control mice, indicating that lnc-HUR1 enhances diethylnitrosamine-induced tumorigenesis. Conclusion: We reveal that HBV-upregulated lnc-HUR1 promotes cell proliferation and tumorigenesis by interacting with p53 to block downstream gene transcription. Our findings suggest that lnc-HUR1 plays an important role in HBV-related hepatocellular carcinoma development and may serve as a therapeutic marker for hepatocellular carcinoma. (Hepatology 2018; 00:000-000).

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“…This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. This strategy led to identify several novel host DENV RBPs by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which were further implicated in DENV replication [185]. …”

Section: Aptamers For Virus Diagnosis and Treatmentmentioning

confidence: 99%

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

González

Martı́n

Fernández³

et al. 2016

Pharmaceuticals

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Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers’ properties as a real tool for viral infection detection and treatment.

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Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Cited by 19 publications

References 46 publications

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Hepatitis B Virus–Upregulated LNC‐HUR1 Promotes Cell Proliferation and Tumorigenesis by Blocking p53 Activity

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

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