Studying RNA-Binding Protein Interactions with Target mRNAs in Eukaryotic Cells: Native Ribonucleoprotein Immunoprecipitation (RIP) Assays

2014 ◽  
pp. 239-246 ◽  
Author(s):  
Joseph A. Cozzitorto ◽  
Masaya Jimbo ◽  
Saswati Chand ◽  
Fernando Blanco ◽  
Shruti Lal ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Eukaryotic Cells ◽  
Target Mrnas

scholarly journals RNA–Binding Protein HuD as a Versatile Factor in Neuronal and Non–Neuronal Systems

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 361
Author(s):  
Myeongwoo Jung ◽  
Eun-Kyung Lee
Keyword(s):  
Gene Expression ◽  
Neural Plasticity ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Novel Treatments ◽  
Target Mrnas ◽  
Neuronal Systems

HuD (also known as ELAVL4) is an RNA–binding protein belonging to the human antigen (Hu) family that regulates stability, translation, splicing, and adenylation of target mRNAs. Unlike ubiquitously distributed HuR, HuD is only expressed in certain types of tissues, mainly in neuronal systems. Numerous studies have shown that HuD plays essential roles in neuronal development, differentiation, neurogenesis, dendritic maturation, neural plasticity, and synaptic transmission by regulating the metabolism of target mRNAs. However, growing evidence suggests that HuD also functions as a pivotal regulator of gene expression in non–neuronal systems and its malfunction is implicated in disease pathogenesis. Comprehensive knowledge of HuD expression, abundance, molecular targets, and regulatory mechanisms will broaden our understanding of its role as a versatile regulator of gene expression, thus enabling novel treatments for diseases with aberrant HuD expression. This review focuses on recent advances investigating the emerging role of HuD, its molecular mechanisms of target gene regulation, and its disease relevance in both neuronal and non–neuronal systems.

scholarly journals Identification and Functional Outcome of mRNAs Associated with RNA-Binding Protein TIA-1

2005 ◽  
Vol 25 (21) ◽  
pp. 9520-9531 ◽  
Author(s):  
Isabel López de Silanes ◽  
Stefanie Galbán ◽  
Jennifer L. Martindale ◽  
Xiaoling Yang ◽  
Krystyna Mazan-Mamczarz ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cellular Damage ◽  
Necrosis Factor Alpha ◽  
Target Mrnas ◽  
Common Motif ◽  
Tnf Α ◽  
Cox 2 ◽  
Rna Complexes

ABSTRACT The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-α) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-α and COX-2 mRNAs and in 3,019 additional UniGene transcripts (∼3% of the UniGene database), localizing preferentially to the 3′ untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.

scholarly journals Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

2021 ◽  
Author(s):  
Sarah E Cabral ◽  
Kimberly Mowry
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Binding Domains ◽  
Multivalent Interactions

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

scholarly journals Erratum: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis

2016 ◽  
Vol 11 (3) ◽  
pp. 616-616 ◽  
Author(s):  
Michael J Moore ◽  
Chaolin Zhang ◽  
Emily Conn Gantman ◽  
Aldo Mele ◽  
Jennifer C Darnell ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals Identification of a signature motif in target mRNAs of RNA-binding protein AUF1

2008 ◽  
Vol 37 (1) ◽  
pp. 204-214 ◽  
Author(s):  
Krystyna Mazan-Mamczarz ◽  
Yuki Kuwano ◽  
Ming Zhan ◽  
Elizabeth J. White ◽  
Jennifer L. Martindale ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Target Mrnas ◽  
Signature Motif

scholarly journals RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

2014 ◽  
Vol 25 (21) ◽  
pp. 3308-3318 ◽  
Author(s):  
Lan Liu ◽  
Eleni Christodoulou-Vafeiadou ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Xiao ◽  
...  
Keyword(s):  
Small Intestine ◽  
Intestinal Epithelium ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Wnt Signaling Pathway ◽  
Small Intestinal Mucosa ◽  
Mucosal Atrophy ◽  
Target Mrnas

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.

scholarly journals Whi3, an S. cerevisiae RNA-Binding Protein, Is a Component of Stress Granules That Regulates Levels of Its Target mRNAs

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e84060 ◽  
Author(s):  
Kristen J. Holmes ◽  
Daniel M. Klass ◽  
Evan L. Guiney ◽  
Martha S. Cyert
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granules ◽  
Target Mrnas

scholarly journals The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs

Cell Reports ◽  
2020 ◽  
Vol 30 (10) ◽  
pp. 3353-3367.e7 ◽  
Author(s):  
John D. Laver ◽  
Jimmy Ly ◽  
Allison K. Winn ◽  
Angelo Karaiskakis ◽  
Sichun Lin ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Target Mrnas ◽  
The Stability
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