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1.
The Evolution and Functional Roles of miR408 and Its Targets in Plants.
Gao, Y, Feng, B, Gao, C, Zhang, H, Wen, F, Tao, L, Fu, G, Xiong, J
International journal of molecular sciences. 2022;(1)
Abstract
MicroRNA408 (miR408) is an ancient and highly conserved miRNA, which is involved in the regulation of plant growth, development and stress response. However, previous research results on the evolution and functional roles of miR408 and its targets are relatively scattered, and there is a lack of a systematic comparison and comprehensive summary of the detailed evolutionary pathways and regulatory mechanisms of miR408 and its targets in plants. Here, we analyzed the evolutionary pathway of miR408 in plants, and summarized the functions of miR408 and its targets in regulating plant growth and development and plant responses to various abiotic and biotic stresses. The evolutionary analysis shows that miR408 is an ancient and highly conserved microRNA, which is widely distributed in different plants. miR408 regulates the growth and development of different plants by down-regulating its targets, encoding blue copper (Cu) proteins, and by transporting Cu to plastocyanin (PC), which affects photosynthesis and ultimately promotes grain yield. In addition, miR408 improves tolerance to stress by down-regulating target genes and enhancing cellular antioxidants, thereby increasing the antioxidant capacity of plants. This review expands and promotes an in-depth understanding of the evolutionary and regulatory roles of miR408 and its targets in plants.
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A comprehensive review of hypomagnesemia.
Ehrenpreis, ED, Jarrouj, G, Meader, R, Wagner, C, Ellis, M
Disease-a-month : DM. 2022;(2):101285
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3.
Ubiquitination-Related miRNA-mRNA Interaction Is a Potential Mechanism in the Progression of Retinoblastoma.
Chen, X, Chen, S, Jiang, Z, Gong, Q, Tang, D, Luo, Q, Liu, X, He, S, He, A, Wu, Y, et al
Investigative ophthalmology & visual science. 2021;(10):3
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Abstract
PURPOSE Retinoblastoma (RB) is the most common primary malignant intraocular cancer. The etiology of RB is complex, and the mechanisms driving its progression remain unclear. Here, we used a series of bioinformatics approaches and experimental methods to investigate the potential regulatory mechanism involved in RB progression. METHODS The common differentially expressed genes were obtained from the public dataset GSE97508. Protein-protein interaction (PPI) network, correlation, and functional enrichment analyses were carried out. The candidate genes were verified in different RB cell lines, and ARPE19 cells served as control. miRNA-mRNA interaction analysis was performed and confirmed by real-time PCR. The CCK-8 assay was conducted to detect cell viability, and the transwell assay was utilized for evaluating the abilities of cell migration and invasion. RESULTS Overall, a total of 258 common differentially expressed genes associated with RB progression were screened out. The PPI network analysis further identified eight downregulated genes mainly enriched in the protein ubiquitination pathway. Moreover, we confirmed UBE2E1, SKP1, FBXO9, FBXO15, and RNF14 from among eight genes through experimental validation in vitro. Furthermore, miRNA-mRNA interaction and real-time PCR analysis of five hub genes revealed that ubiquitination-related miR-548k was involved in RB progression. Loss- and gain-of-function experiments demonstrated that miR-548k and its targets were essential for cell viability, migration, and invasion in the RB cells. CONCLUSIONS Our data indicate that the dysregulation of protein ubiquitination may play an important role in RB progression, and ubiquitination-related miR-548k may be a promising therapeutic target for RB.
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An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation.
Rohner, E, Witman, N, Sohlmer, J, De Genst, E, Louch, WE, Sahara, M, Chien, KR
Molecular medicine (Cambridge, Mass.). 2021;(1):102
Abstract
BACKGROUND The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. METHODS A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. RESULTS Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. CONCLUSION Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.
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In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection.
Ely, A, Singh, P, Smith, TS, Arbuthnot, P
Advanced drug delivery reviews. 2021;:134-146
Abstract
Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.
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HPV DNA genotyping, HPV E6*I mRNA detection, and p16INK4a/Ki-67 staining in Belgian head and neck cancer patient specimens, collected within the HPV-AHEAD study.
Simoens, C, Gorbaslieva, I, Gheit, T, Holzinger, D, Lucas, E, Ridder, R, Rehm, S, Vermeulen, P, Lammens, M, Vanderveken, OM, et al
Cancer epidemiology. 2021;:101925
Abstract
BACKGROUND The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries. METHODS As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC-sites were tested for the presence of type-specific HPV RNA and p16INK4a over-expression. RESULTS The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC. CONCLUSION Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.
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Alternative Polyadenylation of ABC Transporters of the C-Family (ABCC1, ABCC2, ABCC3) and Implications on Posttranscriptional Micro-RNA Regulation.
Bruhn, O, Lindsay, M, Wiebel, F, Kaehler, M, Nagel, I, Böhm, R, Röder, C, Cascorbi, I
Molecular pharmacology. 2020;(2):112-122
Abstract
ATP-binding cassette (ABC) transporters represent a large group of efflux pumps that are strongly involved in the pharmacokinetics of various drugs and nutrient distribution. It was recently shown that micro-RNAs (miRNAs) may significantly alter their expression as proven, e.g., for miR-379 and ABCC2 However, alternative mRNA polyadenylation may result in expression of 3'-untranslated regions (3'-UTRs) with varying lengths. Thus, length variants may result in presence or absence of miRNA binding sites for regulatory miRNAs with consequences on posttranscriptional control. In the present study, we report on 3'-UTR variants of ABCC1, ABCC2, and ABCC3 mRNA. Applying in vitro luciferase reporter gene assays, we show that expression of short ABCC2 3'-UTR variants leads to a significant loss of miR-379/ABCC2 interaction and subsequent upregulation of ABCC2 expression. Furthermore, we show that expression of ABCC2 3'-UTR lengths varies significantly between human healthy tissues but is not directly correlated to the respective protein level in vivo. In conclusion, the presence of altered 3'-UTR lengths in ABC transporters could lead to functional consequences regarding posttranscriptional gene expression, potentially regulated by alternative polyadenylation. Hence, 3'-UTR length variability may be considered as a further mechanism contributing to variability of ABCC transporter expression and subsequent drug variation in drug response. SIGNIFICANCE STATEMENT micro-RNA (miRNA) binding to 3'-untranslated region (3'-UTR) plays an important role in the control of ATP-binding cassette (ABC)-transporter mRNA degradation and translation into proteins. We disclosed various 3'-UTR length variants of ABCC1, C2, and C3 mRNA, with loss of mRNA seed regions partly leading to varying and tissue-dependent interaction with miRNAs, as proven by reporter gene assays. Alternative 3'-UTR lengths may contribute to variable ABCC transporter expression and potentially explains inconsistent findings in miRNA studies.
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In silico and in vitro analysis of cation-activated potassium channels in human corneal endothelial cells.
Amador-Muñoz, D, Gutiérrez, ÁM, Payán-Gómez, C, Matheus, LM
Experimental eye research. 2020;:108114
Abstract
The corneal endothelium is the inner cell monolayer involved in the maintenance of corneal transparence by the generation of homeostatic dehydration. The glycosaminoglycans of the corneal stroma develop a continuous swelling pressure that should be counteracted by the corneal endothelial cells through active transport mechanisms to move the water to the anterior chamber. Protein transporters for sodium (Na+), potassium (K+), chloride (Cl-) and bicarbonate (HCO3-) are involved in this endothelial "pump function", however despite its physiological importance, the efflux mechanism is not completely understood. There is experimental evidence describing transendothelial diffusion of water in the absence of osmotic gradients. Therefore, it is important to get a deeper understanding of alternative models that drive the fluid transport across the endothelium such as the electrochemical gradients. Three transcriptomic datasets of the corneal endothelium were used in this study to analyze the expression of genes that encode proteins that participate in the transport and the reestablishment of the membrane potential across the semipermeable endothelium. Subsequently, the expression of the identified channels was validated in vitro both at mRNA and protein levels. The results of this study provide the first evidence of the expression of KCNN2, KCNN3 and KCNT2 genes in the corneal endothelium. Differences among the level of expression of KCNN2, KCNT2 and KCNN4 genes were found in a differentially expressed gene analysis of the dataset. Taken together these results underscore the potential importance of the ionic channels in the pathophysiology of corneal diseases. Moreover, we elucidate novel mechanisms that might be involved in the pivotal dehydrating function of the endothelium and in others physiologic functions of these cells using in silico pathways analysis.
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Unorthodox Mechanisms to Initiate Translation Open Novel Paths for Gene Expression.
Hernández, G, García, A, Sonenberg, N, Lasko, P
Journal of molecular biology. 2020;(24):166702
Abstract
Translation in eukaryotes is dependent on the activity of translation initiation factor (eIF) 4G family of proteins, a scaffold protein that, during the initiation step, coordinates the activity of other eIFs to recruit the 40S ribosomal subunit to the mRNA. Three decades of research on protein synthesis and its regulation has provided a wealth of evidence supporting the crucial role of cap-dependent translation initiation, which involves eIF4G. However, the recent discovery of a surprising variety of alternative mechanisms to initiate translation in the absence of eIF4G has stirred the orthodox view of how protein synthesis is performed. These mechanisms involve novel interactions among known eIFs, or between known eIFs and other proteins not previously linked to translation. Thus, a new picture is emerging in which the unorthodox translation initiation complexes contribute to the diversity of mechanisms that regulate gene expression in eukaryotes.
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Epitranscriptomics in the Heart: a Focus on m6A.
Longenecker, JZ, Gilbert, CJ, Golubeva, VA, Martens, CR, Accornero, F
Current heart failure reports. 2020;(5):205-212
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PURPOSE OF REVIEW Post-transcriptional modifications are key regulators of gene expression that allow the cell to respond to environmental stimuli. The most abundant internal mRNA modification is N6-methyladenosine (m6A), which has been shown to be involved in the regulation of RNA splicing, localization, translation, and decay. It has also been implicated in a wide range of diseases, and here, we review recent evidence of m6A's involvement in cardiac pathologies and processes. RECENT FINDINGS Studies have primarily relied on gain and loss of function models for the enzymes responsible for adding and removing the m6A modification. Results have revealed a multifaceted role for m6A in the heart's response to myocardial infarction, pressure overload, and ischemia/reperfusion injuries. Genome-wide analyses of mRNAs that are differentially methylated during cardiac stress have highlighted the importance of m6A in regulating the translation of specific categories of transcripts implicated in pathways such as calcium handling, cell growth, autophagy, and adrenergic signaling in cardiomyocytes. Regulation of gene expression by m6A is critical for cardiomyocyte homeostasis and stress responses, suggesting a key role for this modification in cardiac pathophysiology.