scholarly journals Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

2021 ◽  
Author(s):  
Carlos Henrique Vieira-Vieira ◽  
Vita Dauksaite ◽  
Michael Gotthardt ◽  
Matthias Selbach
Keyword(s):  
Posttranslational Modification ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcriptional Level ◽  
Phosphorylation Sites ◽  
Rna Granules ◽  
Regulatory Phosphorylation ◽  
Splicing Regulator ◽  
Regulatory Potential

RNA-binding proteins (RBPs) are major regulators of gene expression at the post- transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.

scholarly journals RNA-binding proteins and circadian rhythms in Arabidopsis thaliana

2001 ◽  
Vol 356 (1415) ◽  
pp. 1755-1759 ◽  
Author(s):  
Dorothee Staiger
Keyword(s):  
Arabidopsis Thaliana ◽  
Feedback Loop ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcriptional Level ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Circadian Oscillators ◽  
Clock Proteins ◽  
Reverse Genetic Approach

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA–binding protein At GRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: At GRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: At grp7 gene transcription is rhythmically activated by the circadian clock during the day. The At GPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, the At GRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the At GRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

scholarly journals Discovery of allele-specific protein-RNA interactions in human transcriptomes

2018 ◽  
Author(s):  
Emad Bahrami-Samani ◽  
Yi Xing
Keyword(s):  
Genetic Variants ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Protein ◽  
Computational Method ◽  
Joint Analysis ◽  
Transcriptional Level ◽  
Rna Seq ◽  
Allele Specific ◽  
Rna Interaction

AbstractGene expression is tightly regulated at the post-transcriptional level through splicing, transport, translation, and decay. RNA-binding proteins (RBPs) play key roles in post-transcriptional gene regulation, and genetic variants that alter RBP-RNA interactions can affect gene products and functions. We developed a computational method ASPRIN (Allele-Specific Protein-RNA Interaction), that uses a joint analysis of CLIP-seq (cross-linking and immunoprecipitation followed by high-throughput sequencing) and RNA-seq data to identify genetic variants that alter RBP-RNA interactions by directly observing the allelic preference of RBP from CLIP-seq experiments as compared to RNA-seq. We used ASPRIN to systematically analyze CLIP-seq and RNA-seq data for 166 RBPs in two ENCODE (Encyclopedia of DNA Elements) cell lines. ASPRIN identified genetic variants that alter RBP-RNA interactions by modifying RBP binding motifs within RNA. Moreover, through an integrative ASPRIN analysis with population-scale RNA-seq data, we showed that ASPRIN can help reveal potential causal variants that affect alternative splicing via allele-specific protein-RNA interactions.

scholarly journals Multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation

2021 ◽  
Author(s):  
Keisuke Hitachi ◽  
Yuri Kiyofuji ◽  
Masashi Nakatani ◽  
Kunihiro Tsuchida
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Myogenic Differentiation ◽  
Cell Physiology ◽  
Transcriptional Level ◽  
Loss Of Function ◽  
Independent Manner ◽  
Multiple Functions

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of hnRNPK for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level via binding to Myoparr. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2α/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays multiple lncRNA-dependent and -independent roles in the inhibition of myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

scholarly journals Nucleocytoplasmic Transport of RNA-Binding Proteins Regulates Neural Stem Cell Fates 

2020 ◽  
Author(s):  
Melissa J. MacPherson ◽  
Sarah L Erickson ◽  
Drayden Kopp ◽  
Pengqiang Wen ◽  
Mohammadreza Aghanoori ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neurodevelopmental Disorder ◽  
Neural Progenitor ◽  
Nucleocytoplasmic Transport ◽  
Transcriptional Level ◽  
Cell Fates ◽  
Cell Fate Decisions

Abstract The formation of the cerebral cortex requires balanced expansion and differentiation of neural progenitor cells, the fate choice of which requires regulation at many steps of gene expression. As progenitor cells often exhibit transcriptomic stochasticity, the ultimate output of cell fate-determining genes must be carefully controlled at the post-transcriptional level, but how this is orchestrated is poorly understood. Here we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb neural progenitor cell fate decisions in mice by disrupting the nucleocytoplasmic transport of CELF2. In self-renewing neural progenitors, CELF2 is localized in the cytoplasm where it binds and coordinates mRNAs that encode cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNAs for translation and thereby triggers neural progenitor differentiation. Our results reveal a mechanism by which transport of CELF2 between discrete subcellular compartments orchestrates an RNA regulon to instruct cell fates in cerebral cortical development.

A Potential Therapeutic Target RNA-binding Protein, Arid5a for the Treatment of Inflammatory Disease Associated with Aberrant Cytokine Expression

2018 ◽  
Vol 24 (16) ◽  
pp. 1766-1771 ◽  
Author(s):  
Kazuya Masuda ◽  
Tadamitsu Kishimoto
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Severe Sepsis ◽  
Inflammatory Cytokines ◽  
Binding Proteins ◽  
Rna Binding ◽  
Cytokine Production ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Transcriptional Level

Background: Infection, tissue damage and aging can cause inflammation with high levels of inflammatory cytokines. Overproduction of inflammatory cytokines often leads to systemic inflammatory response syndrome (SIRS), severe sepsis, and septic shock. However, prominent therapeutic targets have not been found, although the incidence of sepsis is likely to increase annually. Our recent studies indicate that some RNA-binding proteins, which control gene expression of inflammatory cytokines at the post-transcriptional level, may play a critical role in inflammatory diseases such as sepsis. Results: 1) One of the RNA-binding proteins, AT-rich interactive domain-containing 5a (Arid5a) promotes cytokine production through control of mRNA half-lives of pro-inflammatory molecules such as IL-6, STAT3, T-bet, and OX40 in activated macrophages and T cells. Arid5a KO mice are refractory to endotoxin shock, bleomycininduced lung injury, and inflammatory autoimmune disease. 2) Chlorpromazine (CPZ), which is recognized as a psychotic drug, impairs post-transcriptional gene expression of Il6 in LPS-stimulated macrophages: CPZ inhibits the binding activity of Arid5a to the 3’UTR of Il6 mRNA, thereby destabilizing Il6 mRNA possibly through suppression of Arid5a expression. 3) CPZ has strong suppressive effects on cytokine production such as TNF-α in vivo. Mice with treatment of CPZ are resistant to lipopolysaccharide (LPS)-induced shock. Conclusion: Thus, Arid5a contributes to the activation of macrophages and T cells through positive control of mRNA half-lives of inflammatory cytokines and its related molecules, which might lead to cytokine storm. Interestingly, Arid5a was identified from an inhibitory effect of CPZ on IL-6 production in macrophages activated by LPS. Therefore, CPZ derivatives or Arid5a inhibitors may have a potential to suppress severe sepsis through control of post-transcriptional gene expression.

scholarly journals G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress

2020 ◽  
Vol 48 (12) ◽  
pp. 6855-6873 ◽  
Author(s):  
Syam Prakash Somasekharan ◽  
Fan Zhang ◽  
Neetu Saxena ◽  
Jia Ni Huang ◽  
I-Chih Kuo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Stress Adaptation ◽  
Rna Granules ◽  
Before And After ◽  
Selective Translation ◽  
Energy Consuming ◽  
Limit Energy

Abstract Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.

scholarly journals Cataloguing and Selection of mRNAs Localized to Dendrites in Neurons and Regulated by RNA-Binding Proteins in RNA Granules

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 167 ◽  
Author(s):  
Ohashi ◽  
Shiina
Keyword(s):  
Synaptic Plasticity ◽  
Cell Fate ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Memory Formation ◽  
Long Term Memory ◽  
Local Translation ◽  
Rna Granules

Spatiotemporal translational regulation plays a key role in determining cell fate and function. Specifically, in neurons, local translation in dendrites is essential for synaptic plasticity and long-term memory formation. To achieve local translation, RNA-binding proteins in RNA granules regulate target mRNA stability, localization, and translation. To date, mRNAs localized to dendrites have been identified by comprehensive analyses. In addition, mRNAs associated with and regulated by RNA-binding proteins have been identified using various methods in many studies. However, the results obtained from these numerous studies have not been compiled together. In this review, we have catalogued mRNAs that are localized to dendrites and are associated with and regulated by the RNA-binding proteins fragile X mental retardation protein (FMRP), RNA granule protein 105 (RNG105, also known as Caprin1), Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2) in RNA granules. This review provides comprehensive information on dendritic mRNAs, the neuronal functions of mRNA-encoded proteins, the association of dendritic mRNAs with RNA-binding proteins in RNA granules, and the effects of RNA-binding proteins on mRNA regulation. These findings provide insights into the mechanistic basis of protein-synthesis-dependent synaptic plasticity and memory formation and contribute to future efforts to understand the physiological implications of local regulation of dendritic mRNAs in neurons.

scholarly journals Identification of RBPMS as a smooth muscle master splicing regulator via proximity of its gene with super-enhancers

2019 ◽  
Author(s):  
Erick E. Nakagaki-Silva ◽  
Clare Gooding ◽  
Miriam Llorian ◽  
Aishwarya Griselda Jacob ◽  
Frederick Richards ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Control Cell ◽  
Phenotypic Switching ◽  
Regulatory Rna ◽  
Splicing Regulators ◽  
Its Gene ◽  
Splicing Regulator ◽  
Transcription Regulators

AbstractAlternative splicing (AS) programs are primarily controlled by regulatory RNA binding proteins (RBPs). It has been proposed that a small number of master splicing regulators might control cell-specific splicing networks and that these RBPs could be identified by proximity of their genes to transcriptional super-enhancers. Using this approach we identified RBPMS as a critical splicing regulator in differentiated vascular smooth muscle cells (SMCs). RBPMS is highly down-regulated during phenotypic switching of SMCs from a contractile to a motile and proliferative phenotype and is responsible for 20% of the AS changes during this transition. RBPMS directly regulates AS of numerous components of the actin cytoskeleton and focal adhesion machineries whose activity is critical for SMC function in both phenotypes. RBPMS also regulates splicing of other splicing, post-transcriptional and transcription regulators including the key SMC transcription factor Myocardin, thereby matching many of the criteria of a master regulator of AS in SMCs.

scholarly journals Targeting of Post-Transcriptional Regulation as Treatment Strategy in Acute Leukemia

2020 ◽  
Author(s):  
Paulina Podszywalow-Bartnicka ◽  
Magdalena Wolczyk ◽  
Katarzyna Piwocka
Keyword(s):  
Transcriptional Regulation ◽  
Acute Leukemia ◽  
Cancer Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Profile ◽  
Cellular Protein ◽  
Special Focus ◽  
Transcriptional Level ◽  
Post Transcriptional Regulation

Post-transcriptional regulation is an important step of gene expression that allows to fine-tune the cellular protein profile (so called proteome) according to the current demands. That mechanism has been developed to aid survival under stress conditions, however it occurs to be hijacked by cancer cells. Adjustment of the protein profile remodels signaling in cancer cells to adapt to therapeutic treatment, thereby enabling persistence despite unfavorable environment or accumulating mutations. The proteome is shaped at the post-transcriptional level by numerous mechanisms such as alternative splicing, mRNA modifications and triage by RNA binding proteins, change of ribosome composition or signaling, which altogether regulate the translation process. This chapter is an overview of the translation disturbances found in leukemia and their role in development of the disease, with special focus on the possible therapeutic strategies tested in acute leukemia which target elements of those regulatory mechanisms.

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