The scaffolding protein AKAP12 regulates mRNA localization and translation.

Regulation of subcellular messenger (m)RNA localization is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post-transcriptional control of gene expression. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localized activity. Despite the detailed roles of localized mRNAs that have emerged over the past decades, the identity of factors anchoring mRNAs to subcellular domains remains ill-defined. Here, we used an unbiased method to profile the RNA-bound proteome in migrating endothelial cells (ECs) and discovered that the plasma membrane (PM)-associated scaffolding protein A-kinase anchor protein (AKAP)12 interacts with various mRNAs, including transcripts encoding kinases with Actin remodeling activity. In particular, AKAP12 targets a transcript coding for the kinase Abelson Tyrosine-Protein Kinase 2 (ABL2), which we found to be necessary for adequate filopodia formation and angiogenic sprouting. Moreover, we demonstrate that AKAP12 is necessary for anchoring ABL2 mRNA to the PM and show that in the absence of AKAP12, the translation efficiency of ABL2 mRNA is reduced. Altogether, our work identified a unique post-transcriptional function for AKAP12 and sheds light into mechanisms of spatial control of gene expression.

The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2-4EHP (or EIF4E2) translation repressor complex to the mRNA 5'-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifnb1 mRNA that encodes the cytokine interferon ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with argonaute 2 (AGO2), the core component of the miRNA-induced silencing complex (miRISC), via GIGYF2 and enhances the translational repression mediated by natural miRNA-binding sites in the 3' untranslated region of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program by co-opting the host miRNA-mediated silencing machinery.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Mechanisms of impairment of interferon production by SARS-CoV-2.

Interferons (IFNs) are crucial components of the cellular innate immune response to viral infections. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a remarkable capacity to suppress the host IFN production to benefit viral replication and spread. Thus far, of the 28 known virus-encoded proteins, 16 have been found to impair the host's innate immune system at various levels ranging from detection and signaling to transcriptional and post-transcriptional regulation of expression of the components of the cellular antiviral response. Additionally, there is evidence that the viral genome encodes non-protein-coding microRNA-like elements that could also target IFN-stimulated genes. In this brief review, we summarise the current state of knowledge regarding the factors and mechanisms by which SARS-CoV-2 impairs the production of IFNs and thereby dampens the host's innate antiviral immune response.

Gastrointestinal cancer drug resistance: the role of exosomal miRNAs.

Resistance of gastrointestinal (GI) cancer cells to therapeutic agents are one of the major problems in treating this type of cancer. Although the exact mechanism of drug resistance has not yet been fully elucidated, various factors have been identified as contributing factors involved in this process. Several studies have revealed the role of exosomes, especially exosomal microRNAs (miRNAs), in GI tumorigenesis, invasion, angiogenesis, and drug resistance. Exosomes, a type of small extracellular vesicles (EVs), are originated from endosomes and are released into the extracellular environment and body fluids by different cell types. Exosomes mediate cell-cell communication by transferring different cargos, including miRNAs, between parent and recipient cells. Therefore, identifying these exosomal miRNAs and their functions in GI cancers might provide new clues to further explore the secret of this process and thus help in drug-resistance management. This review article will discuss the roles of exosomal miRNAs and their mechanisms of action in drug resistance of different types of GI cancer cells (e.g., stomach, esophagus, liver, pancreas, and colon) to therapeutic agents.

Exosomal noncoding RNAs: key players in glioblastoma drug resistance.

Glioma, as one of the most severe human malignancies, is defined as the Central Nervous System's (CNS) tumors. Glioblastoma (GBM) in this regard, is the most malignant type of gliomas. There are multiple therapeutic strategies to cure GBM, for which chemotherapy is often the first-line treatment. Still, various cellular processes, such as uncontrolled proliferation, invasion and metastasis, may disturb the treatment efficacy. Drug resistance is another process in this way, which can also cause undesirable effects. Thereupon, identifying the mechanisms, involved in developing drug resistance and the relevant mechanisms can be very helpful in GBM management. The discovery of exosomal non-coding RNAs (ncRNAs), RNA molecules that can be transferred between the cells and different tissues using the exosomes, was a milestone in this regard. It has been revealed that the key exosomal ncRNAs, including circular RNAs, microRNAs, and long ncRNAs, are able to modulate GBM drug resistance through different signaling pathways or by affecting regulatory proteins and their corresponding genes. Nowadays, researchers are trying to overcome the limitations of chemotherapy by targeting these RNA molecules. Accordingly, this review aims to clarify the substantial roles of exosomal ncRNAs in GBM drug resistance and involved mechanisms.

Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-ß (IFN-ß). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-ß production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

The intricate balance between microRNA-induced mRNA decay and translational repression.

Post-transcriptional regulation of messenger RNAs (mRNAs) (i.e., mechanisms that control translation, stability and localization) is a critical focal point in spatiotemporal regulation of gene expression in response to changes in environmental conditions. The human genome encodes ~ 2000 microRNAs (miRNAs), each of which could control the expression of hundreds of protein-coding mRNAs by inducing translational repression and/or promoting mRNA decay. While mRNA degradation is a terminal event, translational repression is reversible and can be employed for rapid response to internal or external cues. Recent years have seen significant progress in our understanding of how miRNAs induce degradation or translational repression of the target mRNAs. Here, we review the recent findings that illustrate the cellular machinery that contributes to miRNA-induced silencing, with a focus on the factors that could influence translational repression vs. decay.

Post-Transcriptional Effects of miRNAs on PCSK7 Expression and Function: miR-125a-5p, miR-143-3p, and miR-409-3p as Negative Regulators.

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3'-untranslated region (3'-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in PCSK7 3'-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on PCSK7 expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.

Exosomal noncoding RNAs in prostate cancer.

Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of mortality among men. The recurrent reports of false-positive results of common PCa biomarkers have led to the introduction of some promising biomarkers for PCa, such as exosomal non-coding RNAs (ncRNAs). Exosomes contain various components, such as several ncRNAs (miRNAs and lncRNAs), which are important in the initiation and progression of PCa. These ncRNAs also reflect the state of the origin cell. In this article, we reviewed research on the importance and roles of ncRNAs in PCa, focusing on exosomal ncRNAs. We highlighted plasma exosomal miRNAs (8 miRNAs), urine exosomal miRNAs (19miRNAs), serum miRNAs (2 miRNAs), and five miRNAs in semen used for PCa diagnosis. Also, four exosomal lncRNAs in plasma and urine can be used as biomarkers for PCa diagnosis.

Differential Expression Pattern of linc-ROR Spliced Variants in Pluripotent and Non-Pluripotent Cell Lines.

OBJECTIVE: The human large intergenic non-coding RNA-regulator of reprogramming program (linc-ROR) is known as a stem cell specific linc-RNA. linc-ROR counteracts differentiation via sequestering microRNA-145 (miR-145) that targets OCT4 transcript. Despite the research on the expression and function, the exact structure of linc-ROR transcripts is not clear. Considering the contribution of alternative splicing in transcripts structures and function, identifying different spliced variants of linc-ROR is necessary for further functional analyses. We aimed to find the alternatively spliced transcripts of linc-ROR and investigate their expression pattern in stem and cancer cell lines and during neural differentiation of NT2 cells as a model for understanding linc-ROR role in stem cell and differentiation. MATERIALS AND METHODS: In this experimental study, linc-ROR locus was scanned for identifying novel exons. Different primer sets were used to detect new spliced variants by reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing. Quantitative PCR (qPCR) and RT-PCR were employed to profile expression of linc-ROR transcripts in different cell lines and during neural differentiation of stem cells. RESULTS: We could discover 13 novel spliced variants of linc-ROR harboring unique array of exons. Our work uncovered six novel exons, some of which were the product of exonized transposable elements. Monitoring expression profile of the linc-ROR spliced variants in a panel of pluripotent and non-pluripotent cells exhibited that all transcripts were primarily expressed in pluripotent cells. Moreover, the examined linc-ROR spliced variants showed a similar downregulation during neural differentiation of NT2 cells. CONCLUSION: Altogether, our data showed despite the difference in the structure and composition of exons, various spliced variants of linc-ROR showed similar expression pattern in stem cells and through differentiation.

Multifaceted control of mRNA translation machinery in cancer.

The mRNA translation machinery is tightly regulated through several, at times overlapping, mechanisms that modulate its efficiency and accuracy. Due to their fast rate of growth and metabolism, cancer cells require an excessive amount of mRNA translation and protein synthesis. However, unfavorable conditions, such as hypoxia, amino acid starvation, and oxidative stress, which are abundant in cancer, as well as many anti-cancer treatments inhibit mRNA translation. Cancer cells adapt to the various internal and environmental stresses by employing specialised transcript-specific translation to survive and gain a proliferative advantage. We will highlight the major signaling pathways and mechanisms of translation that regulate the global or mRNA-specific translation in response to the intra- or extra-cellular signals and stresses that are key components in the process of tumourigenesis.

The Role of MicroRNAs in Lung Cancer: Implications for Diagnosis and Therapy.

Lung cancer is the first cause of cancer death in the world due to its high prevalence, aggressiveness, late diagnosis, lack of effective treatment and poor prognosis. It also shows high rate of recurrence, metastasis and drug resistance. All these problems highlight the urgent needs for developing new strategies using noninvasive biomarkers for early detection, metastasis and recurrence of disease. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression post-transcriptionally. These molecules found to be abnormally expressed in increasing number of human disease conditions including cancer. miRNAs could be detected in body fluids such as blood, serum, urine and sputum, which leads us towards the idea of using them as non-invasive biomarker for cancer detection and monitoring cancer treatment and recurrence. miRNAs are found to be deregulated in lung cancer initiation and progression and could regulate lung cancer cell proliferation and invasion. In this review, we summarized recent progress and discoveries in microRNAs regulatory role in lung cancer initiation and progression. In addition, the role of microRNAs in EGFR signaling pathway regulation is discussed briefly.

Circular RNAs and gastrointestinal cancers: Epigenetic regulators with a prognostic and therapeutic role.

Both environmental and genetic factors are involved in the initiation and development of gastrointestinal cancer. Covalent closed circular RNAs (circRNAs) are produced by a mechanism called "back-splicing" from mRNAs. They are highly stable and show cell and tissue specific expression patterns. Although some functions such as "microRNA sponge" and "RNA binding protein sponge" have been reported for a small number of circRNAs, the function of thousands of other circRNAs is still unknown. Dysregulation of circRNAs has been reported in many GI cancers and are involved in metastasis and invasion. CircRNAs have been reported to be useful as prognostic markers and targets for developing new treatments. We first describe the properties and biogenesis of circRNAs. We then summarize recent reports about circRNA functions, expression status, and their potential to be used as biomarkers in GI cancers including, gastric cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, gallbladder cancer and pancreatic cancer.

MicroRNA expression in serum samples of sulfur mustard veterans as a diagnostic gateway to improve care.

Sulfur mustard is a vesicant chemical warfare agent, which has been used during Iraq-Iran-war. Many veterans and civilians still suffer from long-term complications of sulfur mustard exposure, especially in their lung. Although the lung lesions of these patients are similar to Chronic Obstructive Pulmonary Disease (COPD), there are some differences due to different etiology and clinical care. Less is known on the molecular mechanism of sulfur mustard patients and specific treatment options. microRNAs are master regulators of many biological pathways and proofed to be stable surrogate markers in body fluids. Based on that microRNA expression for serum samples of sulfur mustard patients were examined, to establish specific microRNA patterns as a basis for diagnostic use and insight into affected molecular pathways. Patients were categorized based on their long-term complications into three groups and microRNA serum levels were measured. The differentially regulated microRNAs and their corresponding gene targets were identified. Cell cycle arrest, ageing and TGF-beta signaling pathways showed up to be the most deregulated pathways. The candidate microRNA miR-143-3p could be validated on all individual patients. In a ROC analysis miR-143-3p turned out to be a suitable diagnostic biomarker in the mild and severe categories of patients. Further microRNAs which might own a link to the biology of the sulfur mustard patients are miR-365a-3p, miR-200a-3p, miR-663a. miR-148a-3p, which showed up only in a validation study, might be linked to the airway complications of the sulfur mustard patients. All the other candidate microRNAs do not directly link to COPD phenotype or lung complications. In summary the microRNA screening study characterizes several molecular differences in-between the clinical categories of the sulfur mustard exposure groups and established some useful microRNA biomarkers. qPCR raw data is available via the Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110797.

Post-transcriptional Regulation of PCSK9 by miR-191, miR-222, and miR-224.

Since proprotein convertase subtilisin kexin 9 (PCSK9) discovery, a gene involved in LDL metabolism regulation and cardiovascular diseases (CVD), many therapeutic strategies have been introduced for direct targeting of PCSK9. The main goal of these strategies has been to reduce PCSK9 protein level either by application of antibodies or inhibition of its production. In this study, we have tried to discover microRNAs (miRNAs) which can target, and hence regulate, PCSK9 expression. Using bioinformatics tools, we selected three microRNAs with binding sites on 3'-UTR of PCSK9. The expression level of these miRNAs was examined in three different cell lines using real-time RT-PCR. We observed a reciprocal expression pattern between expression level of miR-191, miR-222, and miR-224 with that of PCSK9. Accordingly, the expression levels were highest in Huh7 cells which expressed the lowest level of PCSK9, compared to HepG2 and A549 cell lines. PCSK9 mRNA level also showed a significant decline in HepG2 cells transfected with the vectors overexpressing the aforementioned miRNAs. Furthermore, the miRNAs target sites were cloned in psiCHECK-2 vector, and a direct interaction of the miRNAs and the PCSK9 3'-UTR putative target sites was investigated by means of luciferase assay. Our findings revealed that miR-191, miR-222, and miR-224 can directly interact with PCSK9 3'-UTR and regulate its expression. In conclusion, our data introduces a role for miRNAs to regulate PCSK9 expression.

Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines.

OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4, OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3, is located ∼9kb downstream of the E0 of OCT4C/C1. PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10-24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A, a slight upregulation of OCT4B1, and a dramatic upregulation of OCT4B. Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.