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

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.

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

Open Biology ◽

10.1098/rsob.180110 ◽

2018 ◽

Vol 8 (9) ◽

pp. 180110 ◽

Cited By ~ 2

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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Transcriptome-Wide Identification of RNA Targets of Arabidopsis SERINE/ARGININE-RICH45 Uncovers the Unexpected Roles of This RNA Binding Protein in RNA Processing

The Plant Cell ◽

10.1105/tpc.15.00641 ◽

2015 ◽

Vol 27 (12) ◽

pp. 3294-3308 ◽

Cited By ~ 49

Author(s):

Denghui Xing ◽

Yajun Wang ◽

Michael Hamilton ◽

Asa Ben-Hur ◽

Anireddy S.N. Reddy

Keyword(s):

Rna Processing ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Targets

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RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143

Scientific Reports ◽

10.1038/srep41738 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 24

Author(s):

Hai-Jie Yang ◽

Fei Ju ◽

Xin-Xin Guo ◽

Shuang-Ping Ma ◽

Lei Wang ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Neuroblastoma Cells ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Induced Apoptosis

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

Methods in Molecular Biology - Plant Circadian Networks ◽

10.1007/978-1-4939-0700-7_7 ◽

2014 ◽

pp. 107-121 ◽

Cited By ~ 6

Author(s):

Tino Köster ◽

Meike Haas ◽

Dorothee Staiger

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Targets ◽

Case Identification ◽

Rna Immunoprecipitation

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The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation

International Journal of Molecular Sciences ◽

10.3390/ijms21165633 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5633

Author(s):

Benjamin Lenzen ◽

Thilo Rühle ◽

Marie-Kristin Lehniger ◽

Ayako Okuzaki ◽

Mathias Labs ◽

...

Keyword(s):

Rna Processing ◽

Gene Expression Analysis ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Blot Hybridization ◽

Rna Binding Protein ◽

Target Range ◽

Rna Targets ◽

Chloroplast Rna

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

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

Reproduction Fertility and Development ◽

10.1071/srb10abs149 ◽

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.

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

Brain Sciences ◽

10.3390/brainsci11010085 ◽

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.

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