Decreased mRNA Stability Research Articles

Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2

The various mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a substantial challenge in mitigating the viral infectivity. The identification of novel host factors influencing SARS-CoV-2 replication holds potential for discovering new targets for broad-spectrum antiviral drugs that can combat future viral mutations. In this study, potential host factors regulated by SARS-CoV-2 infection were screened through different high-throughput sequencing techniques and further identified in cells. Subsequent analysis and experiments showed that the reduction of m6A modification level on ACTN4 (Alpha-actinin-4) mRNA leads to a decrease in mRNA stability and translation efficiency, ultimately inhibiting ACTN4 expression. In addition, ACTN4 was demonstrated to target nsp12 for binding and characterized as a competitor for SARS-CoV-2 RNA and the RNA-dependent RNA polymerase complex, thereby impeding viral replication. Furthermore, two ACTN4 agonists, YS-49 and demethyl-coclaurine, were found to dose-dependently inhibit SARS-CoV-2 infection in both Huh7 cells and K18-hACE2 transgenic mice. Collectively, this study unveils the pivotal role of ACTN4 in SARS-CoV-2 infection, offering novel insights into the intricate interplay between the virus and host cells, and reveals two potential candidates for future anti-SARS-CoV-2 drug development.

Abstract Mo054: Long Noncoding RNA LINC00667 Downregulation Leads to SCN5A Alternative Splicing and Arrhythmogenesis

Background: The voltage-gated sodium channel SCN5A, which encodes the Nav1.5 protein, is integral to the propagation of electrical signals in cardiac tissue. SCN5A expression is diminished in cardiomyopathy, significantly increasing the susceptibility to arrhythmias. This reduction in channel activity can be attributed to decreased mRNA stability coupled with aberrant mRNA splicing. The underlying mechanisms driving the dysregulation of mRNA splicing under pathophysiological conditions remain poorly understood. Objective: This study explores the role of long noncoding RNA LINC00667 in the abnormal alternative splicing of SCN5A mRNA. Methods: LINC00667 expression was analyzed using public databases, human heart tissue, iPSC-derived cardiomyocytes (iPSCMs), and cell lines. The impact of SCN5A on action potentials and sodium currents was studied in iPSCMs. LINC00667 and SCN5A expressions were measured using qRT-PCR, while Nav1.5 protein levels were assessed through Western blot. Alternative splicing of SCN5A mRNA was examined using PCR with exon-specific primers. Gene ontology and in silico analysis identified RNA binding proteins (RBPs) potentially interacting with LINC00667. RNA immunoprecipitation (RIP) assays confirmed direct binding with key splice factors. Group differences were assessed using 2-tailed Student’s t-tests, with P < 0.05 indicating significance. Results: LINC00667, found at chromosome 18p11.31, is expressed in the human heart and associated cells, including cardiomyocytes. Its levels are inversely related to arrhythmic risk, decreasing in heart failure and ischemic cardiomyopathy as well as under hypoxic conditions. Gene ontology linked RBPs to SCN5A mRNA splicing, with RBM25, RBM5, and TIA1 identified as LINC00667 interactors. LINC00667 reduction affected regions of SCN5A mRNA encompassing Exons 6, 24, and 28, which could potentially be linked to decreased function/expression or increased unfolded protein response (UPR). Abnormal RNA splicing decreased SCN5A-encoded sodium current, reduced action potential upstroke velocity, and shortened AP duration without altering resting membrane potential. Conclusion: Decreased LINC00667 releases splicing factors that result in abnormal SCN5A mRNA splicing and reduced sodium current. Since LINC00667 is reduced in cardiomyopathy, overexpression may be antiarrhythmic by preventing sodium channel downregulation.

Loss-of-function mutations of the TIE1 receptor tyrosine kinase cause late-onset primary lymphedema.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.

Loss of NAT10 alleviates maternal high-fat diet-induced hepatic steatosis in male offspring of mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is becoming an escalating health problem in pediatric populations. This study aimed to investigate the role of N-acetyltransferase 10 (NAT10) in maternal high-fat diet (HFD)-induced MASLD in offspring at early life. We generated male hepatocyte-specific NAT10 knockout (Nat10HKO) mice and mated them with female Nat10fl/fl mice under chow or HFD feeding. Body weight, liver histopathology, and expression of lipid metabolism-associated genes (Srebp1c, Fasn, Pparα, Cd36, Fatp2, Mttp, and Apob) were assessed in male offspring at weaning. Lipid uptake assays were performed both in vivo and in vitro. The mRNA stability assessment and RNA immunoprecipitation were performed to determine NAT10-regulated target genes. NAT10 deletion in hepatocytes of male offspring alleviated perinatal lipid accumulation induced by maternal HFD, decreasing expression levels of Srebp1c, Fasn, Cd36, Fatp2, Mttp, and Apob while enhancing Pparα expression. Furthermore, Nat10HKO male mice exhibited reduced lipid uptake. In vitro, NAT10 promoted lipid uptake by enhancing the mRNA stability of CD36 and FATP2. RNA immunoprecipitation assays exhibited direct interactions between NAT10 and CD36/FATP2 mRNA. NAT10 deletion in offspring hepatocytes ameliorates maternal HFD-induced hepatic steatosis through decreasing mRNA stability of CD36 and FATP2, highlighting NAT10 as a potential therapeutic target for pediatric MASLD.

M6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

Lithocholic acid decreases mRNA stability of nuclear receptor PPARα by upregulating miR-21 expression in hepatoma HepG2 cells

To investigate the regulatory effects of lithocholic acid (LCA) on nuclear receptor peroxisome proliferatoractivated receptor-alpha (PPARα) mRNA stability at the post-transcriptional level. PPARα 3'UTR luciferase reporter gene vectors were constructed and transfected into HepG2 cells to observe the changes in cellular luciferase activity in response to LCA treatments. Bioinformatic prediction and miRNA PCR array technique were used to identify the differentially expressed miRNAs induced by LCA and their potential binding sites on the 3'UTR. The binding sites (Mut1, Mut2 and Mut1+Mut2) were mutated to compare the changes in cellular luciferase activity following LCA treatment. Western blotting and RTqPCR were used to detect the activated signaling pathway and the expression levels of its downstream transcription factors in LCA-treated cells. The changes in PPARα protein expression level were detected in the cells following overexpression of the transcription factors. Treatment with 100 μmol/L LCA significantly reduced luciferase activity of PPARα 3'UTR1 and 3'UTR2 in HepG2 cells by more than 50% (P<0.01) and induced significant upregulation of miR-21 and miR-22, especially the former (by 2.35 folds, P<0.05). Two predicted miR-21-binding sites in the 3'UTR1 were mutated to construct Mut1, Mut2 and Mut1+Mut2 reporter gene vectors. LCA treatment down-regulated 3'UTR1 luciferase activity by 51%, while Mut1, Mut2, and Mut1+Mut2 were down-regulated by 37%, 39%, and 13%, respectively. LCA caused ERK1/2 phosphorylation and activation of the ERK1/2 signaling pathway, and treatment with 100 μmol/L LCA upregulated the expression of transcription factor early growth response 1 (EGR1) by 5.83 folds (P<0.01). Transient overexpression of EGR1 significantly decreased cellular PPARα protein levels (P<0.05). LCA reduces PPARα mRNA stability and thus decreases PPARα mRNA and protein expressions in hepatocytes by activating the ERK1/2 signaling pathway and upregulating EGR1 and miR-21, which targets 3'UTR regulatory region of PPARα mRNA.

IGF2BP1 enhances the stability of SIK2 mRNA through m6A modification to promote non-small cell lung cancer progression

BackgroundNon-small cell lung cancer (NSCLC) is a significant public health concern globally. Evidence suggests that Salt-inducible kinase 2 (SIK2) is differentially expressed across various cancers and is also implicated in cancer progression. Despite this, the precise function of SIK2 in NSCLC is yet to be elucidated and requires further investigation. MethodsSIK2 expression was evaluated in both HBEC and NSCLC cells, utilizing quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses. Furthermore, to identify the influence of SIK2 on cell proliferation, migration, invasion, and apoptosis, a range of techniques were employed. To evaluate N6-methyladenosine (m6A) modification levels of total RNA and SIK2 within cells, RNA m6A colorimetry and methylated RNA immunoprecipitation (MeRIP) techniques were employed. Additionally, to confirm the interaction between SIK2 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), bioinformatics analysis was executed, and the results were validated through RIP. The stability of SIK2 mRNA was determined using actinomycin D experiment. Furthermore, to validate the in vivo functionality of SIK2, a subcutaneous transplantation tumor model was established in nude mice. ResultsIn this study, upregulation of SIK2 in NSCLC cells was observed. Overexpression of SIK2 was found to lead to promotion of cell proliferation, migration, invasion, and suppression of the Hippo/yes-associated protein (YAP) pathway, while inhibiting apoptosis. RIP analysis showed that IGF2BP1 protein interacted with SIK2 mRNA. Knockdown of IGF2BP1 decreased mRNA stability and m6A modification levels of SIK2. Additionally, knockdown of IGF2BP1 resulted in inhibition of cell proliferation, migration, invasion, suppression of the Hippo/YAP pathway, and promoting apoptosis. Overexpression of SIK2 overturned the impact of IGF2BP1 on NSCLC cells, which was then confirmed through in vivo experiments. ConclusionIGF2BP1 stabilized SIK2 mRNA through m6A modification to promote NSCLC progression, potentially offering new diagnostic and therapeutic insights for NSCLC.

Disruption of CerS6-mediated sphingolipid metabolism by FTO deficiency aggravates ulcerative colitis

Background and aimsDeregulation of RNA N6-methyladenosine (m6A) modification in intestinal epithelial cells (IECs) influences intestinal immune cells and leads to intestinal inflammation. We studied the function of fat mass-and obesity-associated...

Genotoxic carcinogen 7,12-dimethylbenz[a]anthracene inhibits gap junction intercellular communication through post-transcriptional and post-translational processing involved in connexin 43 stability.

Gap junctional intercellular communication (GJIC) is composed of connexin (Cx) and plays an important role in maintaining intracellular homeostasis. Loss of GJIC is involved in the early stages of cancer pathways of non-genotoxic carcinogens; however, the effect of genotoxic carcinogens, including polycyclic aromatic hydrocarbons (PAHs), on GJIC function remains unclear. Therefore, we determined whether and how a representative PAH 7,12-dimethylbenz[a]anthracene (DMBA) suppresses GJIC in WB-F344cells. First, DMBA significantly inhibited GJIC and dose-dependently reduced Cx43 protein and mRNA expression. In contrast, Cx43 promoter activity was upregulated after DMBA treatment via the induction of specificity protein 1 and hepatocyte nuclear factor 3β, indicating that the promoter-independent loss of Cx43 mRNA can be associated with the inhibition of mRNA stability, which was verified by actinomycin D assay. In addition to a decrease in mRNA stability involved in human antigen R, we also observed DMBA-induced acceleration of Cx43 protein degradation, which was closely related to the loss of GJIC through Cx43 phosphorylation via MAPK activation. In conclusion, the genotoxic carcinogen DMBA suppresses GJIC by inhibiting post-transcriptional and post-translational processing of Cx43. Our findings suggest that the GJIC assay is an efficient short-term screening test for predicting the carcinogenic potential of genotoxic carcinogens.

The E3 Ligase TRIM25 Impairs Apoptotic Cell Death in Colon Carcinoma Cells via Destabilization of Caspase-7 mRNA: A Possible Role of hnRNPH1.

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.

11S Proteasome Activator REGγ Promotes Aortic Dissection by Inhibiting RBM3 (RNA Binding Motif Protein 3) Pathway.

Aortic dissection (AD) is a life-threatening cardiovascular disorder with high mortality and lacking underlying mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. Using β-aminopropionitrile (BAPN)-subjected REGγ knockout AD mice and Ang II (angiotensin II)-treated REGγ deficiency vascular smooth muscle cells (VSMCs) to explore the effect of REGγ in AD progression. REGγ was upregulated in mouse aorta of β-aminopropionitrile-induced AD model in vivo and Ang II-treated VSMCs in vitro. REGγ deficiency ameliorated AD progression in β-aminopropionitrile-induced mice by protecting against the switch in VSMCs from contractile to synthetic phenotype through suppressing RBM3 (RNA-binding motif protein 3) decay. Mechanically, REGγ interacted with and degraded the RNA-binding protein RBM3 directly, leading to decreased mRNA stability, lowered expression and transcriptional activity of transcription factor SRF (serum response factor), subsequently reduced transcription of VSMCs-specific contractile genes, α-SMA (alpha-smooth muscle actin) and SM22α (smooth muscle 22 alpha), caused the switch in VSMCs from contractile to synthetic phenotype and associated AD progression. Ablation of endogenous SRF or RBM3, or overexpressing exogenous RBM3 in VSMCs significantly blocked or reestablished the REGγ-dependent action on VSMCs phenotypic switch of Ang II stimulation in vitro. Furthermore, exogenously introducing RBM3 improved the switch in VSMCs from contractile to synthetic phenotype and associated AD features caused by REGγ in vivo. Our results demonstrated that REGγ promoted the switch in VSMCs from contractile to synthetic phenotype and AD progression by inhibiting RBM3-SRF pathway, indicated that modulating REGγ-proteasome activity may be a potential therapeutic approach for AD-associated cardiovascular dysfunction.

PMON56 LH synthesis is regulated by RNA-binding protein ZFP36 bound with AU-rich elements in gonadotropes

Abstract Gonadotropin secretion, which is stimulated by gonadotropin-releasing hormone (GnRH), is a critical feature of reproductive control and maintenance of fertility. GnRH stimulation increases LH secretion 50-100-fold over baseline per pulse, whereas Lhb gene expression increases a modest 1.4-fold. This observation shows that significant post-transcriptional regulation of gonadotropin synthesis and secretion must occur. We have previously shown that GnRH-induced Dusp1 expression, a factor central for feedback control of GnRH signaling in gonadotropes, depends on interaction of the 3'UTR of its mRNA with the RNA binding protein (RBP) ELAVL1, which binds AU-rich elements (ARE) in target mRNAs (1). Recently, using RIP-Chip analysis, we have shown that GnRH receptor (Gnrhr) expression depends on interaction of its mRNA with ELAVL1. Gnrhr mRNA is among the most highly represented ELAVL1-associated mRNAs (2). Further, many mRNAs encoding proteins essential to gonadotropin gene expression, including the immediate-early transcription factor genes Egr1, 2, and 4, Fos, Fosb, Jun, and Junb, as well as Dusp1 and 2, show increased binding to ELAVL1 after GnRH treatment, although gonadotropin Lhb mRNA is not, due to the absence of ARE site to be bound. Typically, association with ELAVL1 stabilize expression of target mRNAs, promoting their translation. Also, of interest is the role of the RBP ZFP36, which competes with ELAVL1 for binding to ARE's but has the opposing effect of directing mRNA to the degradation pathway. Significantly, 5 minutes pulsatile GnRH stimulated ZFP36 protein production in 30-60 minutes in LbetaT2 cells. And, knockdown of ELAVL1 results in the deterioration of Gnrhr expression and of the immediate-early gene Egr1 a transcription factor central to Lhb expression. We have examined the direct role of ZFP36 protein in GnRH-induced gene expression. Interestingly, Gnrhr and Lhb mRNA expression levels were decreased by ZFP36 overexpression in LbetaT2 cells compared with control cells. Also, GnRH-stimulated EGR1 production by was decreased by ZFP36 overexpression. These observations are consistent with previous results with ELAVL1 knockdown showing decreased mRNA stability. We cloned Egr1, Gnrhr and Zfp36 3'UTRs which contain AREs bound by ELAVL1 into luciferase reporter genes in pmirGLO vector to evaluate whether the 3'UTR is directly affected by ZFP36. These Egr1, Gnrhr and Zfp36 3'UTR contained mirGLOs were transfected in both ZFP36 overexpression and control cells and luciferase activity was monitored. The results showed that luciferase expression from reporters bearing the target 3'UTR were significantly reduced in ZFP36 overexpressed cells. Collectively, our studies show that ELAVL1, ZFP36 and their target mRNAs are regulated by GnRH and this indirectly regulates Lhb gene expression in gonadotropes. ) Do MT, T Kim et al. Mol Cell Endocrinology 382 (1): 1346-1357, 2014 ) Terasaka T et al. Endocrinology 160(8): 1999-2014, 2019 Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract P2066: Age-related Changes In Posttranscriptional Regulation Of Macrophage VEGF As A Mechanism Of Impaired Inflammatory Arteriogenesis

Aging and age-related diseases like peripheral arterial disease are associated with impaired inflammatoryarteriogenesis in response to injury. During ischemia, macrophage recruitment and macrophage pro-angiogenic VEGF-A isoform expression contribute to post developmental arteriogenesis. Preliminary datafrom bone marrow-derived macrophages (BMDMs) demonstrated decreased VEGF-A level by both mRNAand protein in aged (52-week-old) and advanced aged (104-week-old) mice when compared to young (12-week-old). Moreover, VEGF-A165a which is VEGF-A proangiogenic splice variant was also reduced inmacrophages from aged and advanced aged mice. The anti-angiogenic VEGF-A165b variant wassignificantly increased in advanced aged mice. We sought to determine the mechanisms which cancontribute to the reduction of pro-angiogenic VEGF-A and consequently reduced inflammatory-mediatedarteriogenesis response in the context of aging. In a hindlimb ischemia model of angio/arteriogenesis, wefound decreased blood flow recovery in aged mice. Moreover, small arterial angiography by microCTconfirmed decreased arteriogenesis. Loss of functional arteriogenesis was associated with decreased muscletissue VEGF-A and VEGF-A165a levels despite adequate macrophage recruitment to the muscle tissue.Moreover, VEGF-A165b was increased in ischemic tissue from advanced aged mice. The mechanism ofreduced macrophage pro-angiogenic VEGF-A165a expression involved decreased mRNA stability withdecreased association of VEGF-A165a mRNA with the RNA-stabilizing protein HuR. In fact, aged BMDMsdemonstrated a loss of HuR binding to VEGF-A165a mRNA with decreased VEGF-A165a mRNA half-life,and consequently decreased inflammatory angio/arteriogenesis.

RNA-binding protein YBX1 promotes brown adipogenesis and thermogenesis via PINK1/PRKN-mediated mitophagy.

Promoting the thermogenic function of brown adipose tissue (BAT) is a promising strategy to combat obesity and metabolic disorders. While much is known about the transcriptional regulation of BAT activation, however, the underlying mechanism of post-transcriptional control by RNA binding proteins remains largely unknown. Here, we found that RNA binding protein Y-box binding protein 1 (YBX1) expression was abundant in BAT and induced by cold exposure and a β-adrenergic agonist in mice. Loss-of-function experiments showed that YBX1 deficiency inhibited mouse primary brown adipocyte differentiation and thermogenic function. Further study showed that YBX1 positively regulates thermogenesis through enhancing mitophagy. Mechanistically, RNA immunoprecipitation identified that YBX1 directly targeted the transcripts of PTEN-induced kinase 1 (Pink1) and parkin RBR E3 ubiquitin-protein ligase (Prkn), two key regulators of mitophagy. RNA decay assay proved that loss of YBX1 decreased mRNA stability of Pink1 and Prkn, leading to reduced protein expression, thereby alleviating mitophagy and inhibiting thermogenic program. Importantly, in vivo experiments demonstrated that YBX1 overexpression in BAT promoted thermogenesis and mitophagy in mice. Collectively, our results reveal novel insight into the molecular mechanism of YBX1 in post-transcriptional regulation of PINK1/PRKN-mediated mitophagy and highlight the critical role of YBX1 in brown adipogenesis and thermogenesis.

Abstract 2368: miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells

Abstract The synchronous activation of BCR and CD40 signaling via B-T cell interactions is required for proliferation of normal (Luo et al, 2018) and some malignant B cells, especially in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). In CLL/FL cells, proliferation occurs mainly in lymph nodes, but not in bone marrow or peripheral blood which lack access to proper B-T cell interactions. Here we have analyzed the mRNA and miRNAs profile in the proliferative intraclonal CLL cell subpopulation that has recently exited lymph node niches (CXCR4dimCD5bright cells; Calissano et al,2011; Pavlasova et al,2016) to reveal molecules potentially participating in synchronous regulation of CD40 and BCR pathway. This has identified 36 miRNAs and 1370 mRNAs differentially expressed in CLL cells exiting lymph nodes as compared to resting non-proliferative CLL cells (CXCR4brightCD5dim cells). Next, we overlapped the 36 miRNAs with their predicted target mRNAs with putative function in CD40/BCR (TargetScan, KEGG) which were anti-correlated to at least one miRNA (miRNAs typically decrease mRNA stability). This revealed among others anti-correlation of lower miR-29 with higher TRAF4 levels in immune niches, which was validated in paired lymph node biopsies vs peripheral blood CLL cells (P&amp;lt;0.01). The negative regulation of TRAF4 by miR-29 was confirmed by transfection of primary CLL and MEC1 cells with synthetic miR-29, miR-29 inhibitor and by a luciferase assay with binding site from TRAF4 3'UTR. Several other miR-29 targets involved in other pathways have been identified as additional targets. TRAF4 is an understudied member of a protein family regulating receptor-induced immune cell activation, however, its role in CD40 pathway or its interactome is unknown. Co-immunoprecipitation and mass spectrometry in B cells pre- and post- stimulation by CD40 ligand revealed TRAF4 interaction with members of the CD40 pathway and a novel function in positive regulation of CD40-induced NFκB activity. In B cells, TRAF4 silencing or its over-expression affected IKKα/β phosphorylation following CD40 ligation or co-culture with activated T cells (all P&amp;lt;0.01). Notably, BCR inhibition lead in vivo to miR-29 upregulation by interfering with its negative regulator MYC induced by BCR. This leads to TRAF4 repression and drastically impairs CD40 signaling (P&amp;lt;0.001), which at least partially explains the observed anti-proliferative effects of BCR inhibitors. Altogether, we have described a novel MYC-miR-29-TRAF4 axis that regulates CD40 signaling in B cells, and acts to synchronize BCR activation with CD40 pathway. Supported by: The ERC under the European Union's Horizon 2020 research and innovation program (grant agreement No 802644), MH CZ, grant nr. NV18-03-00054 and NU20-03-00292. All rights reserved. Czech Science Foundation (20-02566S), MEYS CZ under the project CEITEC 2020 (LQ1601), MH CZ-DRO (FNBr,65269705) and MUNI/A/1595/2020. Citation Format: Sonali Sharma, Vaclav Seda, Eva Vojackova, Gabriela Mladonicka Pavlasova, Daniel Filip, Laura Ondrisova, Veronika Sandova, Lenka Kostalova, Pedro F. Zeni, Marek Borsky, Sarka Pospisilova, Medhat Shehata, Laura Z. Rassenti, Ulrich Jaeger, Michael Doubek, Matthew S. Davids, Jennifer R. Brown, Jiri Mayer, Thomas J. Kipps, Marek Mraz. miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2368.

MicroRNA expression profiling involved in doxorubicin-induced cardiotoxicity using high-throughput deep-sequencing analysis.

MicroRNAs (miRNAs/miRs) are sensitive biomarkers and endogenous repressors of gene expression by decreasing mRNA stability and interfering with mRNA translation. Despite a number of investigations revealing the dysregulation of miRNA expression associated with cardiotoxicity induced by doxorubicin (Dox), perturbation of miRNAs directly resulting from Dox at early stage in cardiomyocytes and the target gene interaction remain largely unknown. In the present study, high-throughput deep-sequencing was used to analyze changes in global miRNA expression in H9c2 cardiomyocytes exposed to 5 µg/ml Dox for 0, 12 or 24 h. Compared with the 0-h time point, the expression levels of 386 unique miRNAs were altered. Based on miRNA expression and fold-change, the target genes of 76 selected miRNAs were further analyzed using gene interaction networks and pathway enrichment analysis. These miRNAs were involved in the regulation of different pathways, whose functions included apoptosis, cell proliferation, extracellular matrix remodeling, oxidative stress and lipid metabolism. These differentially expressed miRNAs included let-7 family, miR-29b-3p, miR-378-3/5p, miR-351-3p, miR-664-3p, miR-455-3p, miR-298-3p, miR-702-5p, miR-128-1-5p, miR-671 and miR-421-5p. The present data indicated that global wide miRNA profiling in Dox-induced cardiomyocytes may provide a novel mechanistic insight into understanding Dox-induced heart failure and cardiotoxicity, as well as novel biomarkers and therapeutic targets.

Genome-wide analysis of lncRNA stability in human.

Transcript stability is associated with many biological processes, and the factors affecting mRNA stability have been extensively studied. However, little is known about the features related to human long noncoding RNA (lncRNA) stability. By inhibiting transcription and collecting samples in 10 time points, genome-wide RNA-seq studies was performed in human lung adenocarcinoma cells (A549) and RNA half-life datasets were constructed. The following observations were obtained. First, the half-life distributions of both lncRNAs and messanger RNAs (mRNAs) with one exon (lnc-human1 and m-human1) were significantly different from those of both lncRNAs and mRNAs with more than one exon (lnc-human2 and m-human2). Furthermore, some factors such as full-length transcript secondary structures played a contrary role in lnc-human1 and m-human2. Second, through the half-life comparisons of nucleus- and cytoplasm-specific and common lncRNAs and mRNAs, lncRNAs (mRNAs) in the nucleus were found to be less stable than those in the cytoplasm, which was derived from transcripts themselves rather than cellular location. Third, kmers-based protein-RNA or RNA-RNA interactions promoted lncRNA stability from lnc-human1 and decreased mRNA stability from m-human2 with high probability. Finally, through applying deep learning-based regression, a non-linear relationship was found to exist between the half-lives of lncRNAs (mRNAs) and related factors. The present study established lncRNA and mRNA half-life regulation networks in the A549 cell line and shed new light on the degradation behaviors of both lncRNAs and mRNAs.

High-throughput 5′P sequencing enables the study of degradation-associated ribosome stalls

SummaryRNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is connected to the translation process up to the degree that 5′-3′ mRNA degradation follows the last translating ribosome. Here, we present an improved high-throughput 5′P degradome RNA-sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable, and uses affordable duplex-specific nuclease-based rRNA depletion. We investigate in vivo ribosome stalls focusing on translation termination. By comparing ribosome stalls identified by ribosome profiling, disome-seq and HT-5Pseq, we find that degradation-associated ribosome stalls are often enriched in Arg preceding the stop codon. On the contrary, mRNAs depleted for those stalls use more frequently a TAA stop codon preceded by hydrophobic amino acids. Finally, we show that termination stalls found by HT-5Pseq, and not by other approaches, are associated with decreased mRNA stability. Our work suggests that ribosome stalls associated with mRNA decay can be easily captured by investigating the 5′P degradome.

IL-10 regulates the malignancy of hemangioma-derived endothelial cells via regulation of PCNA

Hemangioma (HA) is the most common benign tumor and formed by the proliferating endothelial cells of blood vessels. Interleukins (ILs) have been reported to be critical for HA progression. Our present study found that the expression of IL-10 was decreased in HA cells and tissues as compared to their corresponding controls. Treatment with recombinant IL-10 (rIL-10) can suppress the proliferation of HA cells via suppression of proliferating cell nuclear antigen (PCNA), while over expression of PCNA can attenuate rIL-10-inhibited cell proliferation. Further, rIL-10 can decrease the promoter activity and mRNA stability of PCNA in HA cells. Mechanistically, rIL-10 can increase expression of miR-27b-3p to decrease mRNA stability of PCNA, while down regulation of YY1 is involved in rIL-10 suppressed transcription of PCNA. Collectively, IL-10 can suppress the expression of PCNA via miR-27b-3p mediated suppression of mRNA stability and YY1 mediated down regulation of transcription. It suggested that rIL-10 might be a potential therapeutic approach for HA development and progression.

RNAi drugs: Next generation drugs?

Gene therapy aims to apply biotechnological techniques to correct a defective gene through administrating therapeutic nucleic acids or editing a defective gene. The approaches used in gene therapy include traditional transgene, genome editing, such as CRISPR/CAS9, RNA interference (RNAi) and antisense oligonucleotides (ASOs), etc. Gene therapy is promising as a main approach of disease treatment in the future. Particularly, RNAi is a potential rising-star in this field. RNAi is a biological phenomenon in which small RNA molecules inhibit gene expression at post-transcriptional level. There are two categories of RNA molecules, which include small interfering RNAs (siRNAs) and microRNAs (miRNAs). Both RNA types can induce gene silencing through distinct mechanisms, such as degrading mRNA, decreasing mRNA stability, or preventing protein translation. The major difference between siRNAs and miRNAs is that siRNAs are double stranded RNAs with completely paired sequences and high specificity for its targeted mRNA, whereas miRNAs are single stranded RNAs with incomplete complementary sequences and multiple targets on mRNAs. When compared with traditional therapeutic drugs which target defective proteins, siRNA and miRNA drugs offer advantages, such as higher specificity, lower toxicity, and easy manipulation of the defective genes of interest, etc. ASOs technique is another approach to silence a gene at the post-transcriptional level by several mechanisms including recruiting RNase H to cleave and degrade the targeted mRNA, alternative splicing, blocking translation, or binding to miRNA to regulate gene expression. The first-ever RNAi drug was approved in the United States and Europe in 2018, a significant milestone in pharma-cology which represents an era of a new type of drug. This event has been nominated as one of the top ten 2018 scientific breakthroughs. Apparently, it is expected that several other RNAi drugs will be approved for marketing in the near future. The first siRNA drug was used on a rare genetic disease that only affected about 50000 people worldwide. The major obstacle to widely-spread use RNAi drugs is the low delivery efficiency of small RNA molecules into cells or tissues. Various approaches have been developed for RNAi drug delivery in vivo including nanoparticles, cationic lipids, viruses, and polymers. However, they all have limitations for therapeutic applications. The first ASO drug was approved in 1998. Several other ASO drugs were approved in the following twenty years. However, the ASOs drugs have not become a mainstream drug yet due to some limitations other than delivery vehicle, such as adverse side effects from both on-target and off-target toxicities. The RNAi drugs and ASOs drugs may face similar problems in wide clinical applications. The approval of the first RNAi drug will greatly pave a way for the development of more RNAi drugs. We expect current technical hurdles in RNAi delivery will be resolved in the near future and RNAi drugs will become mainstream as next generation drugs.