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RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

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Introduction: ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of hur-silenced RGC in the rat glaucoma model. Methods: The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. Results: Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram. Conclusions: Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR's key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.

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[This corrects the article DOI: 10.3389/fphar.2022.1013098.].

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Pulmonary fibrosis is an irreversible fibrotic process that has a high mortality rate and limited treatment options; thus, developing a novel therapeutic drug is critical. In this study, we synthesized danshensu methyl ester (DME) and explored its anti-pulmonary fibrotic ability on TGF-ß1-stimulated lung fibroblast in vitro and on bleomycin-induced pulmonary fibrosis in vivo. Results showed that DME decreased the expression of differentiation-related proteins, including fibroblast activation protein 1 (FAP1) and S100 calcium-binding protein A4 (S100A4), and fibrotic markers, such as a-SMA, vimentin, and collagen in vivo and in vitro. In addition, DME markedly repressed myofibroblast proliferation and migration. Mechanistically, chromatin immunoprecipitation-PCR, RNA immunoprecipitation, half-life, and other experiments revealed that DME inhibited activating transcription factor 3 expression via TGF-ß1 signal transduction leading to a decrease in lncIAPF transcription and stability. Moreover, DME blocked human antigen R (HuR) nucleocytoplasmic translocation and promoted its degradation via downregulating lncIAPF, which markedly decreased the expression of HuR target genes such as negative autophagic regulators (EZH2, STAT1, and FOXK1). Collectively, our results demonstrated that DME enhanced autophagy to attenuate pulmonary fibrosis via downregulating the lncIAPF-HuR-mediated autophagic axis and the lncIAPF-HuR complex can be the target for drug action.

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BACKGROUND/AIMS: Pre-mRNA splicing is an essential step in eukaryotic gene expression regulation. Genes are composed of exons that remain in the mature mRNAs and intervening sequences named introns. Splicing is the removal of introns and ligation of exons in a mature transcript. Splice site or spliceosome component mutations can lead to different diseases, including neurodegenerative diseases and several cancer types. HuR is an RNA-binding protein that preferentially binds to U- and AU-rich elements, usually found at the 3' UTRs of some mRNAs. We previously observed HuR specifically associated with spliceosomes assembled on introns containing miR-18a and miR-19a. miR-18a and miR-19a are components of the intronic miR-17-92 cluster, along with other five miRNAs. This cluster has been reported to regulate proliferation, migration, and angiogenesis in cells. In this context, we reasoned HuR could be controlling the splicing and processing of these miRNAs, leading to altered cellular phenotypes. METHODS: We induced HuR overexpression in BCPAP and HEK-293T and analyzed the expression of miRNAs using qPCR, as well as the phenotypic effects in those cells. Cell counting to analyze cell growth was performed after trypan blue staining. Migration and invasion assays were performed using transwell filters and cells were counted after staining with crystal violet. We knocked down HuR using a specific siRNA and analyzed expression of miRNAs by qPCR, as well as cellular kinetics. RESULTS: Our results revealed HuR is associated with miR-19a in BCPAP and HEK-293T cells. Conversely, silencing HuR led to reduced miR-17-5p and miR-19a in BCPAP cells. Our data support that HuR stimulates the expression of miR-19, which is further processed and capable of finding its target sequence in a reporter plasmid. Cells overexpressing HuR showed increased cellular proliferation, migration, and invasion rates. Notably, under the presence of antimiR-19a, BCPAP-HuR cells showed reduced cell growth. Taken together, these results indicate the molecular alterations observed are associated with upregulation of miR-19a, leading to cellular processes involved in cancer development. CONCLUSION: Our findings propose a connection between HuR, miRNA biogenesis and cellular modifications. HuR stimulates miR-19a and miR-19b expression, which leads to up-regulation of cell proliferation, migration and invasion, promoting cancer development.

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Chronic stress has been observed to increase the risk of developing depression and induce neuronal alterations of synaptic plasticity, yet the underlying molecular mechanisms remain unclear. Here, we found that the ubiquitously expressed RNA-binding protein HuR was up-regulated in the medial prefrontal cortex (mPFC) of mice following chronic stress. In adult mice, AAV-Cre-mediated knockout of HuR in the mPFC prevented anxiety-like and depression-like behaviors induced by chronic stress. HuR was also required for the stress-induced dendritic spine loss and synaptic transmission deficits. Moreover, HuRflox/flox;Nex-Cre mice, which induce HuR loss of function from embryonic development, exhibited enhanced synaptic functions. Notably, we ascertained RhoA signaling to be regulated by HuR and involved in the modulation of structural synaptic plasticity in response to chronic stress. Our results demonstrate HuR is a critical modulator for the regulation of stress-induced synaptic plasticity alterations and depression, providing a potential therapeutic target for the treatment of depressive disorders.

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Secretion of type I interferon (IFN) is the first cellular reaction to invading pathogens. Despite the protective function of these cytokines, an excessive response to their action can contribute to serious pathologies, such as autoimmune diseases. Transcripts of most cytokines contain adenylate-uridylate (A/U)-rich elements (AREs) that make them highly unstable. RNA-binding proteins (RBPs) are mediators of the regulatory mechanisms that determine the fate of mRNAs containing AREs. Here, we applied an affinity proteomic approach and identified lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR) as the predominant RBP of the IFN-ß mRNA ARE. Reduced expression or chemical inhibition of HuR severely hampered the type I IFN response in various cell lines and fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients. These results define a role for HuR as a potent modulator of the type I IFN response. Taken together, HuR could be used as therapeutic target for diseases where type I IFN production is exaggerated.

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