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

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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Dynamic regulation of immunity through post-translational control of defense transcript splicing

10.1101/736249 ◽

2019 ◽

Author(s):

Keini Dressano ◽

Philipp R Weckwerth ◽

Elly Poretsky ◽

Yohei Takahashi ◽

Carleen Villarreal ◽

...

Keyword(s):

Immune Responses ◽

Translational Control ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Negative Regulator ◽

Signaling Proteins ◽

Receptor Signaling ◽

Post Translational Modification ◽

Defense Signaling

AbstractSurvival of all living organisms requires the ability to detect attack and swiftly counter with protective immune responses. Despite considerable mechanistic advances, interconnectivity of signaling circuits often remains unclear. A newly-characterized protein, IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), negatively regulates immune responses in both maize and Arabidopsis, with disrupted function resulting in enhanced disease resistance. IRR physically interacts with, and promotes canonical splicing of, transcripts encoding defense signaling proteins, including the key negative regulator of pattern recognition receptor signaling complexes, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). Upon immune activation by Plant Elicitor Peptides (Peps), IRR is dephosphorylated, disrupting interaction withCPK28transcripts and resulting in accumulation of an alternative splice variant encoding a truncated CPK28 protein with impaired kinase activity and diminished function as a negative regulator. We demonstrate a novel circuit linking Pep-induced post-translational modification of IRR with post-transcriptionally-mediated attenuation of CPK28 function to dynamically amplify Pep signaling and immune output.One Sentence SummaryPlant innate immunity is promoted by post-translational modification of a novel RNA-binding protein that regulates alternative splicing of transcripts encoding defense signaling proteins to dynamically increase immune receptor signaling capacity through deactivation of a key signal-buffering circuit.

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Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein to Ensure A Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans

10.1101/378398 ◽

2018 ◽

Author(s):

Caroline A. Spike ◽

Gabriela Huelgas-Morales ◽

Tatsuya Tsukamoto ◽

David Greenstein

Keyword(s):

Caenorhabditis Elegans ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Adaptor Protein ◽

Protein Translation ◽

Translational Repression ◽

Meiotic Maturation ◽

Oocyte Growth ◽

Translational Repressor

ABSTRACTIn the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1. Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF-type ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two non-overlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sites is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M-phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte mRNA targets. Based on these observations, we propose a molecular model for the elimination of LIN-41 by SCFSEL-10 and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

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

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02203-2 ◽

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.

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The Nuclear RNA-binding Protein Sam68 Translocates to the Cytoplasm and Associates with the Polysomes in Mouse Spermatocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0548 ◽

2006 ◽

Vol 17 (1) ◽

pp. 14-24 ◽

Cited By ~ 60

Author(s):

Maria Paola Paronetto ◽

Francesca Zalfa ◽

Flavia Botti ◽

Raffaele Geremia ◽

Claudia Bagni ◽

...

Keyword(s):

Translational Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Male Meiosis ◽

Binding Motif ◽

Mitogen Activated Protein ◽

Pachytene Spermatocytes ◽

And Function

Translational control plays a crucial role during gametogenesis in organisms as different as worms and mammals. Mouse knockout models have highlighted the essential function of many RNA-binding proteins during spermatogenesis. Herein we have investigated the expression and function during mammalian male meiosis of Sam68, an RNA-binding protein implicated in several aspects of RNA metabolism. Sam68 expression and localization within the cells is stage specific: it is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. During the meiotic divisions, Sam68 translocates to the cytoplasm where it is found associated with the polysomes. Translocation correlates with serine/threonine phosphorylation and it is blocked by inhibitors of the mitogen activated protein kinases ERK1/2 and of the maturation promoting factor cyclinB-cdc2 complex. Both kinases associate with Sam68 in pachytene spermatocytes and phosphorylate the regulatory regions upstream and downstream of the Sam68 RNA-binding motif. Molecular cloning of the mRNAs associated with Sam68 in mouse spermatocytes reveals a subset of genes that might be posttranscriptionally regulated by this RNA-binding protein during spermatogenesis. We also demonstrate that Sam68 shuttles between the nucleus and the cytoplasm in secondary spermatocytes, suggesting that it may promote translation of specific RNA targets during the meiotic divisions.

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Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.1023 ◽

1995 ◽

Vol 129 (4) ◽

pp. 1023-1032 ◽

Cited By ~ 67

Author(s):

J M Schumacher ◽

K Lee ◽

S Edelhoff ◽

R E Braun

Keyword(s):

Translational Control ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Translational Repression ◽

Rna Transport ◽

Binding Domains ◽

Cytoplasmic Microtubules ◽

Pachytene Spermatocytes ◽

Expression Libraries

Previous studies in transgenic mice have established the importance of the 3' untranslated region (UTR) of the spermatid-specific protamine-1 (Prm-1) mRNA in its translational control during male germ cell development. To clone genes that mediate the translational repression or activation of the Prm-1 mRNA, we screened cDNA expression libraries made with RNA from pachytene spermatocytes and round spermatids, with an RNA probe corresponding to the 3' UTR of Prm-1. We obtained six independent clones that encode Spnr, a spermatid perinuclear RNA-binding protein. Spnr is a 71-kD protein that contains two previously described RNA binding domains. The Spnr mRNA is expressed at high levels in the testis, ovary, and brain, and is present in multiple forms in those tissues. Immunolocalization of the Spnr protein within the testis shows that it is expressed exclusively in postmeiotic germ cells and that it is localized to the manchette, a spermatid-specific microtubular array. Although the Spnr protein is expressed too late to be directly involved in the translational repression of Prm-1 specifically, we suggest that the Spnr protein may be involved in other aspects of spermatid RNA metabolism, such as RNA transport or translational activation.

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Cbk1 Regulation of the RNA-Binding Protein Ssd1 Integrates Cell Fate with Translational Control

Current Biology ◽

10.1016/j.cub.2009.10.071 ◽

2009 ◽

Vol 19 (24) ◽

pp. 2114-2120 ◽

Cited By ~ 62

Author(s):

Jaclyn M. Jansen ◽

Antony G. Wanless ◽

Christopher W. Seidel ◽

Eric L. Weiss

Keyword(s):

Cell Fate ◽

Translational Control ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein

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144. ROLE OF RNA-BINDING PROTEIN, MUSASHI-1 (Msi-1), IN MURINE FOLLICULOGENESIS AND OOCYTE DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/srb09abs144 ◽

2009 ◽

Vol 21 (9) ◽

pp. 62

Author(s):

K. M. Gunter ◽

B. A. Fraser ◽

A. P. Sobinoff ◽

N. A. Siddall ◽

G. R. Hime ◽

...

Keyword(s):

Real Time ◽

Oocyte Maturation ◽

Real Time Pcr ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Mrna Translation ◽

Oocyte Development ◽

Target Mrna ◽

P21 Waf1

Follicular development and oocyte maturation in mammals requires the temporal and spatial control of protein production. Consequently, it is hypothesised that the preovulatory follicle represses mRNA translation until specific proteins are required during oocyte maturation. Increasingly RNA-binding proteins are being recognised as important contributors to germ cell development, particularly during oocyte transcriptional quiescence. We have identified the presence of RNA-binding protein musashi-1 (Msi-1) mRNA within the mouse ovary and mature mouse oocyte, where the protein is believed to act as a translational repressor by binding to specific sequences within the 3' UTR of target mRNA molecules. Recent studies in various mammalian systems have identified p21 WAF1, cdkn2a, notch and m-numb as potential targets of Msi-1. We have also identified morf4l1 as a potential target through preliminary pulldown and microarray analysis using a GST tagged Msi-1 recombinant protein. To further study these potential targets, a transgenic Msi-1 mouse was produced to overexpress the RNA-binding protein in the developing oocyte. Real time PCR, performed on intact ovaries of WT and Tg mice, has so far demonstrated a 1.5-fold increase in Msi-1 expression in tgMsi-1/+ ovaries, above WT ovary expression. Real time PCR analysis of Msi-1 target mRNA expression has also shown an overall increase in expression in the tgMsi-1/+ ovaries of p21 WAF1 (~2.5-fold), cdkn2a (~2-fold), and notch (~3-fold). However m-numb and morf4l1 do not appear to be targets of Msi-1 in the oocyte, with no significant difference in expression between the WT and tgMsi-1/+ ovaries analysed. Functional quantification of oocyte development reveals a significantly less oocytes produced from superovulated juvenile mice compared with wild type litter mates. Therefore, preliminary analysis suggests that Msi-1 may play a role in binding the transcripts of genes necessary for cell cycle regulation and chromatin remodelling, characteristic of meiotic progression and oocyte development.

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