RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Torres, Manon; Becquet, Denis; Guillen, S; Boyer, Bénédicte; Moreno, M.; Blanchard, Marie-Pierre; Franc, Jean‐Louis; François‐Bellan, Anne‐Marie

doi:10.3791/57379-v

Cited by 10 publications

(9 citation statements)

References 5 publications

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“…To explore the RNA interactome of Tug1, a previously established RNA pull-down protocol for long non-coding RNAs (lncRNAs) was employed 14 . The secondary structure of lncRNA Tug1 was predicted using RNAstructure Webserver, and an antisense biotinylated DNA oligonucleotide probe with minimal probability of internal base pairing was designed via the Fold algorithm 15 .…”

Section: Methodsmentioning

confidence: 99%

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

Koka,

Li,

Akther

et al. 2023

Preprint

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Friedreich's Ataxia (FRDA) is a neurodegenerative disorder caused by reduced frataxin (FXN) levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia with no cure, FRDA lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study identifies long non-coding RNA Tug1 as a potential blood-based FRDA biomarker. In a previous study using a frataxin knockdown mouse model (FRDAkd), we observed several hallmark FRDA symptoms and abnormalities in various tissues. Building on this, we hypothesized that a dual-source approach-comparing the data from peripheral blood samples from FRDA patients with tissue samples from affected areas in FRDAkd mice, tissues usually unattainable from patients-would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of FRDA patients, carriers, and controls, as well as 192 tissue datasets from FRDAkd mice. Blood and tissue samples underwent RNA isolation and qRT-PCR, and frataxin knockdown was confirmed through ELISA. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum and blood samples on an independent set of 45 healthy controls, 45 FRDA patients; 66 heterozygous carriers, and 72 FRDA patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the FRDAkd mouse model (One-way ANOVA, p ≤ 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R2 = 0.71 during depletion, R2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1's downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of FRDA patients compared to controls (Wilcoxon signed-rank test, p < 0.05). Regression analyses revealed a negative correlation between TUG1 levels and disease onset (p < 0.0037), and positive correlations with disease duration and Functional Disability Stage score (p < 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. In summary, this study highlights Tug1 as a crucial blood-based biomarker for FRDA. Tug1's consistent expression variance across human and mouse tissues is closely associated to disease severity and key FRDA pathways. It also correlates strongly with Fxn levels, making it a promising early, non-invasive marker. TUG1 offers potential for FRDA monitoring and therapeutic development, warranting further clinical research.

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Section: Methodsmentioning

confidence: 99%

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

Koka,

Li,

Akther

et al. 2023

Preprint

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“…RNA pull-down was performed according to previous reports with minor modifications ( 18 , 19 ). Briefly, rat DRGs were fixed with 1 ml of 1% paraformaldehyde solution for 10 min and homogenized in 500 µl of proteinase K solution (100 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 0.5% SDS, and 200 U/ml RNase OUT).…”

Section: Methodsmentioning

confidence: 99%

“…One milliliter of hybridization buffer (700 mM NaCl, 70 mM Tris-HCl, 0.1 mM EDTA, 1.25% SDS, and 200 U/ml RNase OUT and 15% formamide) was added and 20 µl of samples were collected as input samples. Neat1-specific (5′-GCCTTCCCACATTTAAAAACACAAC-3′) or negative control (5′-TAAAATACCATTTGATGTTTGAAATTAT-3′) oligonucleotide probes ( 18 ) biotinylated at the 3′-end (100 pmol) were added and incubated for 4 h at room temperature with agitation in a rotator. Magnetic beads (Dynabeads MyOne Streptavidin C1, Thermo Fisher Scientific) were added and incubated at room temperature overnight with agitation.…”

Section: Methodsmentioning

confidence: 99%

Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury

et al. 2023

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Primary sensory neurons regulate inflammatory processes in innervated regions through neuro-immune communication. However, how their immune-modulating functions are regulated in concert remains largely unknown. Here, we show that Neat1 long non-coding RNA (lncRNA) organizes the proinflammatory gene expressions in the dorsal root ganglion (DRG) in chronic intractable neuropathic pain in rats. Neat1 was abundantly expressed in the DRG and was upregulated after peripheral nerve injury. Neat1 overexpression in primary sensory neurons caused mechanical and thermal hypersensitivity, whereas its knockdown alleviated neuropathic pain. Bioinformatics analysis of comprehensive transcriptome changes indicated the inflammatory response was the most relevant function of genes upregulated through Neat1. Consistent with this, upregulation of proinflammatory genes in the DRG following nerve injury was suppressed by Neat1 knockdown. Expression changes of these proinflammatory genes were regulated through Neat1-mRNA interaction-dependent and -independent mechanisms. Notably, Neat1 increased proinflammatory genes by stabilizing its interacting mRNAs in neuropathic pain. Finally, Neat1 in primary sensory neurons contributed to spinal inflammatory processes that mediated peripheral neuropathic pain. These findings demonstrate that Neat1 lncRNA is a key regulator of neuro-immune communication in neuropathic pain.

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“…The RNA pull-down assay was conducted using the Magnetic RNA-Protein Pull-Down kit (20164, thormo scientific) according to the instructions of the manufacturer. 22 The biotin-labeled circ_0003596 probe, the miR-502-5p probe and the corresponding antisense probe were purchased from GenePharma (China, Shanghai), which were 5 0 -ATCCCTGGAGCATCCTTGGTTAG-3. The isolation of RNA pull-down from the biotinylated probes was amplified through the PCR.…”

Section: Rna Pull-down Assaymentioning

confidence: 99%

Hsa_circ_0003596 enhances the development of cell renal clear cell carcinoma through the miR‐502‐5p/IGF1/PI3K/AKT axis

Fang

Liang

Dong

et al. 2023

The Journal of Gene Medicine

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Accumulating research findings have shown that circular RNAs (circRNAs) play an indispensable role in tumorigenesis and tumor progression. The current study aimed to explore the role and modulatory mechanism of hsa_circ_0003596 in clear cell renal cell carcinoma (ccRCC). Quantitative real‐time polymerase chain reaction was adopted to detect the expression of hsa_circ_0003596 in ccRCC tissue and cell lines. 5‐Ethynyl‐2′‐deoxyuridine, cell counting kit 8 and the colony formation assay were utilized to assess the proliferation potential of the ccRCC cells. Transwell along with wound healing assays were adopted to quantify infiltration coupled with the migration potential of the cells. The current research study found that the circRNA hsa_circ_0003596 was overexpressed in ccRCC tissue and cell lines. Further, result showed that hsa_circ_0003596 was associated with distant metastasis of renal cancer. Notably, the knockdown of hsa_circ_0003596 can lower the proliferation, infiltration and migration potential of ccRCC cells. The results of in vivo experiments found that the reduction of hsa_circ_0003596 significantly hampered the growth of tumors in mice. In addition, it was evident that hsa_circ_0003596 acts as a “molecular sponge” for miR‐502‐5p to upregulate the expression of the microRNA‐502‐5p (miR‐502‐5p) target insulin‐like growth factor 1 (IGF1R). Furthermore, it was found that the phosphatidylinositol 3‐kinase (PI3K)/AKT signaling was the downstream cascade of hsa_circ_0003596/miR‐502‐5p/IGF1R cascade, which is partly responsible for the cancer‐promoting effect. Overall, the results of the present study showed that hsa_circ_0003596 facilitated the proliferation, infiltration and migration of ccRCC through the miR‐502‐5p/IGF1R/PI3K/AKT axis. Therefore, it was evident that hsa_circ_0003596 can serve as a possible biomarker and therapeutic target against ccRCC.

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RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Cited by 10 publications

References 5 publications

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

Long non-coding RNA TUG1 is down-regulated in Friedreich’s ataxia

Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury

Hsa_circ_0003596 enhances the development of cell renal clear cell carcinoma through the miR‐502‐5p/IGF1/PI3K/AKT axis

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