scholarly journals Rapid Deadenylation Triggered by a Nonsense Codon Precedes Decay of the RNA Body in a Mammalian Cytoplasmic Nonsense-Mediated Decay Pathway

2003 ◽  
Vol 23 (14) ◽  
pp. 4805-4813 ◽  
Author(s):  
Chyi-Ying A. Chen ◽  
Ann-Bin Shyu
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Decay Kinetics ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Exonuclease Digestion

ABSTRACT Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5′-to-3′ exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.

scholarly journals Human DICER helicase domain recruits PKR and modulates its antiviral activity

2021 ◽  
Vol 17 (5) ◽  
pp. e1009549
Author(s):  
Thomas C. Montavon ◽  
Morgane Baldaccini ◽  
Mathieu Lefèvre ◽  
Erika Girardi ◽  
Béatrice Chane-Woon-Ming ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Rna Binding ◽  
Semliki Forest Virus ◽  
Rna Binding Proteins ◽  
Helicase Domain ◽  
Type I ◽  
Dependent Manner ◽  
Rna Helicases

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.

scholarly journals RNA dependent suppression of C9orf72 ALS/FTD associated neurodegeneration by Matrin-3

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Nandini Ramesh ◽  
Elizabeth L. Daley ◽  
Amanda M. Gleixner ◽  
Jacob R. Mann ◽  
Sukhleen Kour ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Matrin 3 ◽  
Rna Foci ◽  
Ran Translation

Abstract The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.

scholarly journals Rab1b and ARF5 are novel RNA-binding proteins involved in IRES-driven RNA localization

2018 ◽  
Author(s):  
Javier Fernandez-Chamorro ◽  
Rosario Francisco-Velilla ◽  
Jorge Ramajo ◽  
Encarnación Martinez-Salas
Keyword(s):  
Binding Proteins ◽  
Protein Transport ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Domain 3

ABSTRACTInternal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here we have combined proteomic and imaging analysis to study novel IRES interactors recognizing specific RNA structural subdomains. Besides known IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, these proteins exert different effects on translation. While a dominant-negative mutant of Rab1b decreased IRES function, ARF5 silencing stimulated IRES activity. RNA FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctuated distribution. Second, the IRES-driven RNA colocalized with ARF5 and Rab1b, but not with the dominant-negative of Rab1b. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

scholarly journals miRNAmotif—A Tool for the Prediction of Pre-miRNA–Protein Interactions

2018 ◽  
Vol 19 (12) ◽  
pp. 4075 ◽  
Author(s):  
Martyna Urbanek-Trzeciak ◽  
Edyta Jaworska ◽  
Wlodzimierz Krzyzosiak
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Web Server ◽  
Mature Mirnas ◽  
Mirna Biogenesis ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Protein Motifs ◽  
The Web

MicroRNAs (miRNAs) are short, non-coding post-transcriptional gene regulators. In mammalian cells, mature miRNAs are produced from primary precursors (pri-miRNAs) using canonical protein machinery, which includes Drosha/DGCR8 and Dicer, or the non-canonical mirtron pathway. In plant cells, mature miRNAs are excised from pri-miRNAs by the DICER-LIKE1 (DCL1) protein complex. The involvement of multiple regulatory proteins that bind directly to distinct miRNA precursors in a sequence- or structure-dependent manner adds to the complexity of the miRNA maturation process. Here, we present a web server that enables searches for miRNA precursors that can be recognized by diverse RNA-binding proteins based on known sequence motifs to facilitate the identification of other proteins involved in miRNA biogenesis. The database used by the web server contains known human, murine, and Arabidopsis thaliana pre-miRNAs. The web server can also be used to predict new RNA-binding protein motifs based on a list of user-provided sequences. We show examples of miRNAmotif applications, presenting precursors that contain motifs recognized by Lin28, MCPIP1, and DGCR8 and predicting motifs within pre-miRNA precursors that are recognized by two DEAD-box helicases—DDX1 and DDX17. miRNAmotif is released as an open-source software under the MIT License. The code is available at GitHub (www.github.com/martynaut/mirnamotif). The webserver is freely available at http://mirnamotif.ibch.poznan.pl.

scholarly journals C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

2021 ◽  
Author(s):  
Eun Seon Kim ◽  
Chang Geon Chung ◽  
Jeong Hyang Park ◽  
Byung Su Ko ◽  
Sung Soon Park ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Heterozygous Mutation ◽  
Pathological Feature ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Drosophila Model ◽  
Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

scholarly journals Targeting Poison Exons to Treat Developmental and Epileptic Encephalopathy

2021 ◽  
pp. 1-6
Author(s):  
Miriam C. Aziz ◽  
Patricia N. Schneider ◽  
Gemma L. Carvill
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Autism Spectrum ◽  
Epileptic Encephalopathy ◽  
Nonsense Mediated Decay ◽  
Subsequent Degradation ◽  
Alternative Splicing Events ◽  
Genetic Epilepsies

Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic <i>SCN1A</i> variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

scholarly journals RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome

2018 ◽  
Vol 293 (43) ◽  
pp. 16596-16607 ◽  
Author(s):  
Jackson B. Trotman ◽  
Bernice A. Agana ◽  
Andrew J. Giltmier ◽  
Vicki H. Wysocki ◽  
Daniel R. Schoenberg
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Capping Enzyme ◽  
Eukaryotic Mrnas

The N7-methylguanosine cap is added in the nucleus early in gene transcription and is a defining feature of eukaryotic mRNAs. Mammalian cells also possess cytoplasmic machinery for restoring the cap at uncapped or partially degraded RNA 5′ ends. Central to both pathways is capping enzyme (CE) (RNA guanylyltransferase and 5′-phosphatase (RNGTT)), a bifunctional, nuclear and cytoplasmic enzyme. CE is recruited to the cytoplasmic capping complex by binding of a C-terminal proline-rich sequence to the third Src homology 3 (SH3) domain of NCK adapter protein 1 (NCK1). To gain broader insight into the cellular context of cytoplasmic recapping, here we identified the protein interactome of cytoplasmic CE in human U2OS cells through two complementary approaches: chemical cross-linking and recovery with cytoplasmic CE and protein screening with proximity-dependent biotin identification (BioID). This strategy unexpectedly identified 66 proteins, 52 of which are RNA-binding proteins. We found that CE interacts with several of these proteins independently of RNA, mediated by sequences within its N-terminal triphosphatase domain, and we present a model describing how CE-binding proteins may function in defining recapping targets. This analysis also revealed that CE is a client protein of heat shock protein 90 (HSP90). Nuclear and cytoplasmic CEs were exquisitely sensitive to inhibition of HSP90, with both forms declining significantly following treatment with each of several HSP90 inhibitors. Importantly, steady-state levels of capped mRNAs decreased in cells treated with the HSP90 inhibitor geldanamycin, raising the possibility that the cytotoxic effect of these drugs may partially be due to a general reduction in translatable mRNAs.

scholarly journals Neurogenesis: Regulation by Alternative Splicing and Related Posttranscriptional Processes

2016 ◽  
Vol 23 (5) ◽  
pp. 466-477 ◽  
Author(s):  
Enrique Lara-Pezzi ◽  
Manuel Desco ◽  
Alberto Gatto ◽  
María Victoria Gómez-Gaviro
Keyword(s):  
Alternative Splicing ◽  
Protein Function ◽  
Posttranscriptional Regulation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Degradation ◽  
Protein Isoforms ◽  
Mammalian Brain ◽  
Nonsense Mediated Decay

The complexity of the mammalian brain requires highly specialized protein function and diversity. As neurons differentiate and the neuronal circuitry is established, several mRNAs undergo alternative splicing and other posttranscriptional changes that expand the variety of protein isoforms produced. Recent advances are beginning to shed light on the molecular mechanisms that regulate isoform switching during neurogenesis and the role played by specific RNA binding proteins in this process. Neurogenesis and neuronal wiring were recently shown to also be regulated by RNA degradation through nonsense-mediated decay. An additional layer of regulatory complexity in these biological processes is the interplay between alternative splicing and long noncoding RNAs. Dysregulation of posttranscriptional regulation results in defective neuronal differentiation and/or synaptic connections that lead to neurodevelopmental and psychiatric disorders.

scholarly journals Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4505
Author(s):  
Hilde Sundvold
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Low Density Lipoprotein ◽  
Ring Finger ◽  
Density Lipoprotein ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Human Hepatoma Cells ◽  
Density Lipoprotein Receptor ◽  
The Stability

An increased understanding of low-density lipoprotein receptor (LDLR) and its regulation may facilitate drug development for the treatment of hypercholesterolemia. Triciribine (TCN), which is a highly selective AKT inhibitor, increases the stability of LDLR mRNA downstream of extracellular signal-regulated kinase (ERK) in human hepatoma cells (HepG2). Here, a candidate approach was used in order to determine whether the RNA-binding proteins (RBPs) ZFP36 ring finger protein like 1 (ZFP36L1) and Hu antigen R (HuR) play a role in TCN-mediated stabilization of LDLR mRNA. The depletion of HuR led to a reduction of LDLR mRNA stability, an event that was more pronounced in TCN-treated cells. TCN was found to induce the translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ZFP36L1 depletion increased the stability of LDLR mRNA consistent with its destabilizing role. However, in contrast to HuR, TCN had no effect on LDLR mRNA turnover in ZFP36L1-depleted cells. TCN induced the phosphorylation of ZFP36L1 in an ERK/RSK-dependent manner and promoted its dissociation from the CCR4-NOT complex. In sum, these data suggest that TCN utilizes ERK signaling to increase the activity of HuR and inhibit ZFP36L1 to stabilize LDLR mRNA in HepG2 cells.

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