The RIPper Case: Identification of RNA-Binding Protein Targets by RNA Immunoprecipitation

2014 ◽  
pp. 107-121 ◽  
Author(s):  
Tino Köster ◽  
Meike Haas ◽  
Dorothee Staiger
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Targets ◽  
Case Identification ◽  
Rna Immunoprecipitation

scholarly journals RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Kausik Bishayee ◽  
Khadija Habib ◽  
Uddin Md. Nazim ◽  
Jieun Kang ◽  
Aniko Szabo ◽  
...  
Keyword(s):  
Cancer Survival ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Neuroblastoma Cells ◽  
Cancer Type ◽  
Rna Targets ◽  
Stress Survival ◽  
Promote Cell Survival ◽  
Rna Immunoprecipitation

Abstract Background Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. Methods RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. Results In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. Conclusions HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.

scholarly journals A deep learning framework for modeling structural features of RNA-binding protein targets

2015 ◽  
Vol 44 (4) ◽  
pp. e32-e32 ◽  
Author(s):  
Sai Zhang ◽  
Jingtian Zhou ◽  
Hailin Hu ◽  
Haipeng Gong ◽  
Ligong Chen ◽  
...  
Keyword(s):  
Deep Learning ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Structural Features ◽  
Protein Targets ◽  
Learning Framework

149. TRANSLATIONAL CONTROL IN FOLLICULOGENESIS AND OOCYTE DEVELOPMENT: A ROLE FOR RNA-BINDING PROTEIN MUSASHI-1

2010 ◽  
Vol 22 (9) ◽  
pp. 67
Author(s):  
K. M. Gunter ◽  
B. A. Fraser ◽  
A. P. Sobinoff ◽  
V. Pye ◽  
N. A. Siddall ◽  
...  
Keyword(s):  
Translational Control ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Oocyte Development ◽  
Cell Cycle Regulators ◽  
Translational Repressor ◽  
Qpcr Analysis ◽  
Mrna Pool ◽  
Rna Immunoprecipitation

Control of the maternal mRNA pool during oocyte maturation is crucial to the correct temporal and spatial expression of proteins, particularly during oocyte transcriptional quiescence. We have identified Musashi-1 as being present within the oocyte/ovary, where this RNA-binding protein is believed to act as a translational repressor of target mRNAs. Recent studies in mammalian neural and intestinal systems have identified a number of cell cycle regulators as potential targets of Msi-1. Using Msi-1 protein-RNA immunoprecipitation, we have also identified musashi-2 (msi-2) and c-mos as putative targets in the mouse oocyte. To further study these targets, a transgenic mouse was produced to overexpress Msi-1 exclusively in the oocyte. QPCR analysis, performed on intact ovaries of wild type (WT) and Tg mice, confirmed a 1.5-fold increase in msi-1 expression in tgMsi-1/+ ovaries in excess of WT ovary expression. QPCR analysis of Msi-1 target expression, performed on intact WT and Tg ovaries, in conjunction with transcript obtained from the Msi-1 protein-RNA immunoprecipitation, revealed an overall increase in expression in the tgMsi-1/+ and Msi-1 IP samples, respectively, of p21WAF-1 (~2.5-fold; undetected), cdkn2a (~2-fold; undetected), notch1 (~3-fold;undetected), c-mos (no difference; ~41-fold) and msi-2 (~7-fold; ~10-fold). Immunohistochemical analysis of Msi-2 protein expression in transgenic juvenile mouse ovaries,demonstrated a decrease in expression of Msi-2 in tgMsi-1/+ ovaries, when compared to WT ovary expression, suggesting that Msi-2 mRNA is translationally repressed by Msi-1. Therefore, preliminary analysis suggests that Msi-1 may play a role inregulating transcripts of genes necessary for processes characteristic of meiotic progression and oocyte development.

scholarly journals AEG-1 Regulates TWIK-1 Expression as an RNA-Binding Protein in Astrocytes

2021 ◽  
Vol 11 (1) ◽  
pp. 85
Author(s):  
Hyun-Gug Jung ◽  
Ajung Kim ◽  
Seung-Chan Kim ◽  
Jae-Yong Park ◽  
Eun Mi Hwang
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Expression Patterns ◽  
Rna Binding Protein ◽  
Brain Diseases ◽  
Real Time Quantitative Pcr ◽  
Cultured Astrocytes ◽  
Rna Immunoprecipitation ◽  
Mrna And Protein Expression

AEG-1, also called MTDH, has oncogenic potential in numerous cancers and is considered a multifunctional modulator because of its involvement in developmental processes and inflammatory and degenerative brain diseases. However, the role of AEG-1 in astrocytes remains unknown. This study aimed to investigate proteins directly regulated by AEG-1 by analyzing their RNA expression patterns in astrocytes transfected with scramble shRNA and AEG-1 shRNA. AEG-1 knockdown down-regulated TWIK-1 mRNA. Real-time quantitative PCR (qPCR) and immunocytochemistry assays confirmed that AEG-1 modulates TWIK-1 mRNA and protein expression. Electrophysiological experiments further revealed that AEG-1 further regulates TWIK-1-mediated potassium currents in normal astrocytes. An RNA immunoprecipitation assay to determine how AEG-1 regulates the expression of TWIK-1 revealed that AEG-1 binds directly to TWIK-1 mRNA. Furthermore, TWIK-1 mRNA stability was significantly increased upon overexpression of AEG-1 in cultured astrocytes (p < 0.01). Our findings show that AEG-1 serves as an RNA-binding protein to regulate TWIK-1 expression in normal astrocytes.

Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development

2007 ◽  
Vol 75 (6) ◽  
pp. 519-528 ◽  
Author(s):  
Hye-Won Song ◽  
Karen Cauffman ◽  
Agnes P. Chan ◽  
Yi Zhou ◽  
Mary Lou King ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Meiotic Maturation ◽  
Early Cleavage ◽  
Germline Development ◽  
Protein Targets

scholarly journals Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

2017 ◽  
Author(s):  
Weijin Xu ◽  
Reazur Rahman ◽  
Michael Rosbash
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Target Identification ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Targets ◽  
Rna Targets ◽  
Editing Activity ◽  
Editing Enzyme

AbstractWe previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR and performs Adenosine-to-Inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

scholarly journals The RNA-binding protein GRSF1 promotes hepatocarcinogenesis via competitively binding to YY1 mRNA with miR-30e-5p

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Lili Han ◽  
Chen Huang ◽  
Xiaofei Wang ◽  
Dongdong Tong
Keyword(s):  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Xenograft Model ◽  
Interaction Network ◽  
Rna Immunoprecipitation ◽  
Small Molecule Compound ◽  
Underlying Mechanisms

Abstract Background Dysregulation of RNA binding protein (RBP) expression has been confirmed to be causally linked with tumorigenesis. The detailed biological effect and underlying mechanisms of the RBP GRSF1 in hepatocellular carcinoma (HCC) remain unclear. Methods HCC cells with stable knockdown of GRSF1 were established using two sh-RNA-encoding lentiviruses. The functions of GRSF1 in HCC were explored using MTT, colony formation, flow cytometry, and Transwell assays and a xenograft model. Transcriptomic sequencing in GRSF1-deficient MHCC-97H cells was carried out to identify the downstream effector of GRSF1. The regulatory mechanisms among GRSF1, YY1 and miR-30e-5p were investigated via RNA immunoprecipitation, luciferase, RNA pull-down and ChIP assays. Several in vivo assays were used to assess the selectivity of the small-molecule compound VE-821 in HCC and to confirm the absence of general toxicity in animal models. Results GRSF1 was frequently increased in HCC tissue and cells and was associated with worse clinical outcomes. GRSF1 functions as a novel oncogenic RBP by enhancing YY1 mRNA stability, and the GUUU motifs within the YY1 3`UTR 2663-2847 were the specific binding motifs for GRSF1. YY1 feedback promoted GRSF1 expression by binding to the GRSF1 promoter. In addition, YY1 was a critical target of miR-30e-5p, which was confirmed in this study to inhibit HCC hepatocarcinogenesis. GRSF1 and miR-30e-5p competitively regulated YY1 by binding to its 3`UTR 2663-2847 region. Finally, we identified that VE-821 blocked HCC progression by inhibiting the GRSF1/YY1 pathway. Conclusion This study revealed the interaction network among GRSF1, YY1 and miR-30e-5p, providing new insight into HCC pathogenesis, and indicated that VE821 may serve as a novel agent with potential for HCC treatment through inhibition of the GRSF1/YY1 axis.

Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP) v1

protocols.io ◽  
2020 ◽  
Author(s):  
Eric L. Van Nostrand ◽  
Thai B. Nguyen ◽  
Chelsea Gelboin-Burkhart ◽  
Ruth Wang ◽  
Steven M. Blue ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cost Effective ◽  
Protein Targets

scholarly journals mmi1 and rep2 mRNAs are novel RNA targets of the Mei2 RNA-binding protein during early meiosis in Schizosaccharomyces pombe

Open Biology ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 180110 ◽  
Author(s):  
Kaustav Mukherjee ◽  
Bruce Futcher ◽  
Janet Leatherwood
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rna Binding ◽  
Binding Protein ◽  
Mrna Level ◽  
Rna Binding Protein ◽  
S Phase ◽  
Non Coding Rna ◽  
Rna Targets ◽  
Rna Immunoprecipitation ◽  
Meiotic Initiation

The RNA-binding protein Mei2 is crucial for meiosis in Schizosaccharomyces pombe. In mei2 mutants, pre-meiotic S-phase is blocked, along with meiosis. Mei2 binds a long non-coding RNA (lncRNA) called meiRNA, which is a ‘sponge RNA’ for the meiotic inhibitor protein Mmi1. The interaction between Mei2, meiRNA and Mmi1 protein is essential for meiosis. But mei2 mutants have stronger and different phenotypes than meiRNA mutants, since mei2Δ arrests before pre-meiotic S, while the meiRNA mutant arrests after pre-meiotic S but before meiosis. This suggests Mei2 may bind additional RNAs. To identify novel RNA targets of Mei2, which might explain how Mei2 regulates pre-meiotic S, we used RNA immunoprecipitation and cross-linking immunoprecipitation. In addition to meiRNA, we found the mRNAs for mmi1 (which encodes Mmi1) and for the S-phase transcription factor rep2 . There were also three other RNAs of uncertain relevance. We suggest that at meiotic initiation, Mei2 may sequester rep2 mRNA to help allow pre-meiotic S, and then may bind both meiRNA and mmi1 mRNA to inactivate Mmi1 at two levels, the protein level (as previously known), and also the mRNA level, allowing meiosis. We call Mei2–meiRNA a ‘double sponge’ (i.e. binding both an mRNA and its encoded protein).

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