Circular RNA CircPDS5B impairs angiogenesis following ischemic stroke through its interaction with hnRNPL to inactivate VEGF-A.

BACKGROUND: Ischemic stroke (IS) is the primary cause of mortality and disability worldwide. Circular RNAs (circRNAs) have been proposed as crucial regulators in IS. This study focused on the role of circPDS5B in IS and its underlying mechanism. METHOD: Transient middle cerebral artery occlusion (tMCAO) mice and glucose deprivation/reoxygenation (OGD/R)-exposed human brain microvascular endothelial cells (BMECs) were used as IS models. Expression levels of circPDS5B, heterogenous nuclear ribonucleoprotein L (hnRNPL), runt-related transcription factor-1 (Runx1), and Zinc finger protein 24 (ZNF24) were quantified by qRT-PCR. MTT, wound healing, transwell and tube formation assays were employed to evaluate the cell proliferation, migration, and angiogenesis, respectively. Moreover, RNA pull-down, and RIP assay were performed to investigate the interaction among circPDS5B, hnRNPL and vascular endothelial growth factor-A (VEGF-A). RESULTS: circPDS5B was significantly up-regulated in IS patients and tMCAO mice. Deficiency of circPDS5B relieved brain infarction and neuronal injury of tMCAO mice. OGD/R-induced apoptosis, inhibition in viability, migration, and angiogenesis in BMECs were dramatically abrogated by circPDS5B knockdown. Mechanistically, circPDS5B stabilized Runx1 and ZNF24 via recruiting hnRNPL, thereby suppressing the transcription and expression of VEGFA. hnRNPL silencing strengthened circPDS5B knockdown-mediated beneficial effect on IS. CONCLUSION: Altogether, our study showed that high expression of circPDS5B exacerbated IS through recruitment of hnRNPL to stabilize Runx1/ZNF24 and subsequently inactivate VEGFA. Our findings suggest circPDS5B may be a novel therapeutic target for IS.

The SWI/SNF subunit BRG1 affects alternative splicing by changing RNA binding factor interactions with nascent RNA.

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.

LINC01588 regulates WWP2-mediated cardiomyocyte injury by interacting with HNRNPL.

Cardiomyocyte dysfunction and apoptosis induced by ischemia-hypoxia are common features of many acute and chronic heart diseases. WW domain-containing E3 ubiquitin ligase (WWP2) has been identified as an important regulator in pathogenesis of some health-threatening diseases. Although a couple of recent reports prompted the potential role of WWP2 in heart dysfunction, however, its exact role and how its expression was regulated in ischemic-hypoxic cardiomyocytes are still elusive. Here, we found that WWP2 protein level was induced in anoxia/reoxygenation (A/R) treated cardiomyocytes in a time-dependent manner, accompanied by synchronous expression of LINC01588 and HNRNPL. Knockdown of LINC01588 increased cardiomyocyte apoptosis, the level of oxidative stress, and expression of pro-inflammatory cytokine genes, down-regulated the expression of WWP2 and promoted expression of SEPT4 gene that contributed to cardiomyocyte dysfunction and was a target gene of WWP2. LINC01588 overexpression improved the functions of A/R treated cardiomyocytes, up-regulated WWP2 and reduced SEPT4 expression. In the mechanism exploration, we found that LINC01588 could directly bind with HNRNPL protein that could interact with WWP2, suggesting that WWP2 was involved in the regulation of LINC01588 in A/R treated cardiomyocytes. Moreover, WWP2 inhibition declined the protective role of LINC01588 in cardiomyocyte dysfunction induced by A/R. Finally, we demonstrated that LINC01588 overexpression improved acute myocardial infarction in mice in vivo. In conclusion, LINC01588 improved A/R-induced cardiomyocyte dysfunction by interacting with HNRNPL and promoting WWP2-mediated degradation of SEPT4.

Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

Long noncoding RNA LEF1-AS1 binds with HNRNPL to boost the proliferation, migration, and invasion in osteosarcoma by enhancing the mRNA stability of LEF1.

Osteosarcoma (OS) is the most frequent type of cancer that starts in the bones, with a rather high tendency to metastasize to other bones at the early stages. Although many types of research have demonstrated that long noncoding RNAs commonly take part in the development of various cancers, the modulating mechanism of LEF1-AS1 in OS was unknown yet. In this study, our results disclosed that LEF1-AS1, as well as LEF1, had higher expression levels in OS cells than that in normal bone cells. LEF1-AS1 knockdown dramatically inhibited the proliferation, migration, as well as invasion in OS, which proved that LEF1-AS1 contributed to the growth of OS. Furthermore, HNRNPL knockdown suppressed the expression of LEF1. LEF1-AS1 was confirmed to sponge HNRNPL and HNRNPL could bind with LEF1. Both LEF1-AS1 and HNRNPL could enhance the stability of LEF1 mRNA. LEF1-AS1 acted as a promoter in stimulating the Wnt signaling pathway in OS. In rescue experiments, overexpression of LEF1 partially offset the inhibition LEF1-AS1 knockdown brought in the proliferation, migration as well as invasion of OS cells. Collectively, this study had investigated that LEF1-AS1 bound with HNRNPL to promote OS cell proliferation, migration as well as invasion by enhancing the messenger RNA stability of LEF1.

HnRNP L represses cryptic exons.

The fidelity of RNA splicing is regulated by a network of splicing enhancers and repressors, although the rules that govern this process are not yet fully understood. One mechanism that contributes to splicing fidelity is the repression of nonconserved cryptic exons by splicing factors that recognize dinucleotide repeats. We previously identified that TDP-43 and PTBP1/PTBP2 are capable of repressing cryptic exons utilizing UG and CU repeats, respectively. Here we demonstrate that hnRNP L (HNRNPL) also represses cryptic exons by utilizing exonic CA repeats, particularly near the 5'SS. We hypothesize that hnRNP L regulates CA repeat repression for both cryptic exon repression and developmental processes such as T cell differentiation.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing.

Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

HnRNPL inhibits the osteogenic differentiation of PDLCs stimulated by SrCl2 through repressing Setd2.

Osteoporosis has been shown to intensify bone loss caused by periodontitis and both share common risk factors. One strategy utilized to manage the disease has been via the release of Sr ions by Strontium Ranelate having a direct effect on preventing osteoclast activation and promoting osteoblast differentiation. Previously we have developed and characterized porous Sr-mesoporous bioactive glass (Sr-MBG) scaffolds and demonstrated their ability to promote periodontal regeneration when compared to MBG alone. Our group further discovered a splicing factor, heterogeneous nuclear ribonucleoprotein L (hnRNPL), was drastically down-regulated in periodontal ligament stem cells (PDLCs) stimulated by Sr through the activation of AKT pathway. Furthermore, hnRNPL restrained the osteogenic differentiation of PDLCs through down-regulating H3K36me3-specific methyltransferase Setd2. The goal of the present study was to investigate the mechanism of periodontal regeneration stimulated by Sr It was first found that the epigenetic mechanism of splicing factor hnRNPL participated in the osteogenesis processing of PDLCs stimulated by SrCl2 . Meanwhile, the different role of hnRNPL and SET domain containing 2 (Setd2) may provide some implication of the treatment of periodontitis patients simultaneously suffering from osteoporosis.

LINC02273 drives breast cancer metastasis by epigenetically increasing AGR2 transcription.

BACKGROUND: The majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown. METHODS: lncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment. RESULTS: We identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo. CONCLUSIONS: Our findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention.

Inhibition of nonsense-mediated RNA decay by ER stress.

Nonsense-mediated RNA decay (NMD) selectively degrades mutated and aberrantly processed transcripts that contain premature termination codons (PTC). Cellular NMD activity is typically assessed using exogenous PTC-containing reporters. We overcame some inherently problematic aspects of assaying endogenous targets and developed a broadly applicable strategy to reliably and easily monitor changes in cellular NMD activity. Our new method was genetically validated for distinguishing NMD regulation from transcriptional control and alternative splicing regulation, and unexpectedly disclosed a different sensitivity of NMD targets to NMD inhibition. Applying this robust method for screening, we identified NMD-inhibiting stressors but also found that NMD inactivation was not universal to cellular stresses. The high sensitivity and broad dynamic range of our method revealed a strong correlation between NMD inhibition, endoplasmic reticulum (ER) stress, and polysome disassembly upon thapsigargin treatment in a temporal and dose-dependent manner. We found little evidence of calcium signaling mediating thapsigargin-induced NMD inhibition. Instead, we discovered that of the three unfolded protein response (UPR) pathways activated by thapsigargin, mainly protein kinase RNA-like endoplasmic reticulum kinase (PERK) was required for NMD inhibition. Finally, we showed that ER stress compounded TDP-43 depletion in the up-regulation of NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis and frontotemporal dementia, and that the additive effect of ER stress was completely blocked by PERK deficiency.

Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical ß1α1ß2ß3α2ß4 topology, except for an unusual fifth ß-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two ß-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the ß-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.

BRCA1 exon 11 a model of long exon splicing regulation.

BRCA1 exon 11 is one of the biggest human exons, spanning 3426 bases. This gene is potentially involved in DNA repair as well as cell growth and cell cycle control. Exon 11 is regulated at the splicing level producing three main different combinations of BRCA1 mature transcripts; one including the whole of exon 11 (full isoform), one skipping the entire exon (D11 isoform), and one including only 117 base pairs of exon 11 (D11q isoform). Using minigene and deletion analyses, we have previously described important splicing regulatory sequences located at the beginning of this exon (5' end). We have now found additional important sequences located at its 3' end. In particular, we describe the presence of a strong splicing enhancer adjacent to the downstream 5' splice site, which minimizes competition from an upstream 5' splice site and so ensures long exon inclusion. Analyses of the proteins binding these RNA sequences have revealed that Tra2beta and hnRNP L are involved in the regulation of BRCA1 exon 11 by influencing the recognition of donor sites. Interestingly, BRCA1 exon 11 carrying deletion of the regulatory sequences bound by these factors also showed unexpected responses to up- or downregulation of these regulatory proteins, suggesting that they can also bind elsewhere in this large exon and elicit different effects on its recognition.   The identification of sequences and proteins relevant for the regulation of BRCA1 exon 11 now provides better knowledge on how this exon is recognized and may represent an important step toward understanding how large exons are regulated.

HNRNPL Increases WSB1 mRNA Stability to Promote Proliferation and Lipid Droplets in Clear Cell Renal Cell Carcinoma.

This study aims to explore the possible effect and mechanism of heterogeneous nuclear ribonucleoprotein L (HNRNPL) on the lipid droplet and proliferation ability of clear cell renal cell carcinoma (ccRCC). The mRNA and protein expressions of HNRNPL and WSB1 on ccRCC tissues and cells were detected using qRT-PCR and western blot. The lipid droplet of cells was assessed after Oil Red O staining and BODIPY 493/503 staining. Cell proliferation was detected by CCK-8 assay. The interaction between HNRNPL and WSB1 was verified using RNA immunoprecipitation (RIP) and RNA-pull down assay. WSB1 mRNA stability was measured by Actinomycin D. Elevated expressions of HNRNPL and WSB1 were found in both ccRCC tissues and cells. HNRNPL knockdown can lead to suppressed lipid droplet and cell proliferation ability of ccRCC cells, while expression pattern was found in cells with HNRNPL overexpression. RIP and RNA-pull down assay clarified the binding of HNRNPL with WSB1. HNRNPL can facilitate the stability and expression of WSB1 mRNA. Rescue assay identified the promotive effect of HNRNPL on lipid droplets and cell proliferation of ccRCC cells can be abolished in response to WSB1 knockdown. Collected evidence summarized that HNRNPL can increase the stability of WSB1 mRNA to promote lipid droplet and proliferation ability in ccRCC cells.

HNRNPL facilitates ferroptosis in hepatocellular carcinoma cells by promoting S100A9 expression.

OBJECTIVE: This study probed into the effect of HNRNPL on ferroptosis in hepatocellular carcinoma (HCC) cells and related molecular mechanisms. METHODS: Expression patterns of HNRNPL, Recombinant S100 Calcium Binding Protein A9 (S100A9) were analyzed in HCC tissues or cells. Following transfection, HCC cell activity was analyzed, followed by detection of levels of ROS, iron content, LPO, MDA, and GSH as well as the expression of ferroptosis-related proteins. For molecular mechanism, RIP, RNA pull-down assay and actinomycin D assay were implemented to verify the binding relationship between HNRNPL and S100A9. Finally, in vivo nude mouse xenograft tumor experiments were performed for further validate the crucial role of HNENPL expression in HCC. RESULTS: HNRNPL and S100A9 were significantly overexpressed in HCC. sh-HNRNPL treatment led to a significant decrease in cellular activity, GSH content, and expression of GPX4 and SLC7A11, and a significant increase in iron content, LPO level, MDA, ROS content, and expression of ACSL4 and TFR1. In addition, after sh-HNRNPL was combined with oe-S100A9 or Fer-1, a ferroptosis inhibitor, both oe-S100A9 and Fer-1 reversed the promotional effect of sh-HNRNPL on ferroptosis of HCC cells when sh-HNRNPL acted alone. Mechanically, HNRNPL promoted S100A9 mRNA stability and expression through RBP. Furthermore, low expression of HNRNPL in vivo delayed the growth of xenograft tumors and the expression of ferroptosis-related proteins. CONCLUSION: HNRNPL promotes S100A9 mRNA stability and expression through RBP action, thereby promoting ferroptosis in HCC cells.

hnRNPL expression dynamics in the embryo and placenta.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay.

Background: CFTR nonsense alleles generate negligible CFTR protein due to the nonsense mutation: 1) triggering CFTR mRNA degradation by nonsense-mediated mRNA decay (NMD), and 2) terminating CFTR mRNA translation prematurely. Thus, people with cystic fibrosis (PwCF) who carry nonsense alleles cannot benefit from current modulator drugs, which target CFTR protein. In this study, we examined whether PTBP1 and HNRNPL, two RNA binding proteins that protect a subset of mRNAs with a long 3' untranslated region (UTR) from NMD, similarly affect CFTR mRNA.Silencing RNAs were used to deplete PTBP1 or HNRNPL in 16HBE14o- human bronchial epithelial cells expressing WT, G542X, or W1282X CFTR. CFTR mRNA abundance was measured relative to controls by quantitative PCR. PTBP1 and HNRNPL were also exogenously expressed in each cell line and CFTR mRNA levels were similarly quantified. Results: PTBP1 depletion reduced CFTR mRNA abundance in all three 16HBE14o- cell lines; HRNPL depletion reduced CFTR mRNA abundance in only the G542X and W1282X cell lines. Notably, decreased CFTR mRNA abundance correlated with increased mRNA decay. Exogenous expression of PTBP1 or HNRNPL increased CFTR mRNA abundance in all three cell lines; HNRNPL overexpression generally increased CFTR to a greater extent in G542X and W1282X 16HBE14o- cells.Our data indicate that PTBP1 and HNRNPL regulate CFTR mRNA abundance by protecting CFTR transcripts from NMD. This suggests that PTBP1 and/or HNRNPL may represent potential therapeutic targets to increase CFTR mRNA abundance and enhance responses to CFTR modulators and other therapeutic approaches in PwCF.

Dnmt3bas coordinates transcriptional induction and alternative exon inclusion to promote catalytically active Dnmt3b expression.

Embryonic expression of DNMT3B is critical for establishing de novo DNA methylation. This study uncovers the mechanism through which the promoter-associated long non-coding RNA (lncRNA) Dnmt3bas controls the induction and alternative splicing of Dnmt3b during embryonic stem cell (ESC) differentiation. Dnmt3bas recruits the PRC2 (polycomb repressive complex 2) at cis-regulatory elements of the Dnmt3b gene expressed at a basal level. Correspondingly, Dnmt3bas knockdown enhances Dnmt3b transcriptional induction, whereas overexpression of Dnmt3bas dampens it. Dnmt3b induction coincides with exon inclusion, switching the predominant isoform from the inactive Dnmt3b6 to the active Dnmt3b1. Intriguingly, overexpressing Dnmt3bas further enhances the Dnmt3b1:Dnmt3b6 ratio, attributed to its interaction with hnRNPL (heterogeneous nuclear ribonucleoprotein L), a splicing factor that promotes exon inclusion. Our data suggest that Dnmt3bas coordinates alternative splicing and transcriptional induction of Dnmt3b by facilitating the hnRNPL and RNA polymerase II (RNA Pol II) interaction at the Dnmt3b promoter. This dual mechanism precisely regulates the expression of catalytically active DNMT3B, ensuring fidelity and specificity of de novo DNA methylation.

Long non-coding RNA lincRNA-erythroid prosurvival attenuates inflammation by enhancing myosin heavy chain 6 stability through recruitment of heterogeneous nuclear ribonucleoprotein L in myocardial infarction.

Myocardial infarction (MI), a prevalent cardiac disorder with high mortality, leads to severe heart injury associated with inflammation and cardiomyocyte apoptosis. Long non-coding RNAs have been widely found to participate in the progression of MI. Here, we aimed to explore the impact of lincRNA-erythroid prosurvival (EPS) on MI-induced inflammation and cardiomyocyte apoptosis. Significantly, lincRNA-EPS was lowly expressed in MI mice and in oxygen and glucose deprivation (OGD)-treated HL-1 cells. Echocardiography analysis revealed that lincRNA-EPS overexpression increased left ventricular ejection fraction and left ventricular fraction shortening, and decreased left ventricular internal diameter at end systole and left ventricular internal diameter at end diastole in a mouse model. In our study, the expression levels of interleukin-6, tumor necrosis factor-alpha, interleukin-1ß, and interleukin-18 were upregulated in the MI mice and OGD-treated HL-1 cells, while lincRNA-EPS overexpression reversed these phenotypes. Meanwhile, lincRNA-EPS reduced MI-induced cardiomyocyte apoptosis in vivo and in vitro. Mechanically, lincRNA-EPS interacted with myosin heavy chain 6 (MYH6) and heterogeneous nuclear ribonucleoprotein L (HNRNPL), and the depletion of lincRNA-EPS and HNRNPL inhibited MYH6 mRNA stability in HL-1 cells. HNRNPL knockdown blocked lincRNA-EPS overexpression-induced MYH6 expression in the system. The depletion of MYH6 and HNRNPL could rescue lincRNA-EPS overexpression-reduced inflammation and apoptosis in HL-1 cells. Thus, we conclude that lincRNA-EPS attenuates inflammation and apoptosis in MI-induced myocardial injury by maintaining MYH6 stability through the recruitment of HNRNPL.

Long non-coding RNA lincRNA-erythroid prosurvival (EPS) alleviates cerebral ischemia/reperfusion injury by maintaining high-temperature requirement protein A1 (Htra1) stability through recruiting heterogeneous nuclear ribonucleoprotein L (HNRNPL).

This study aimed at investigating the role and mechanism of lincRNA-EPS (erythroid prosurvival) in cerebral ischemia/reperfusion (CIR) injury. The results showed that the overexpression of lincRNA-EPS was able to reduce the levels of interleukin-6, tumor necrosis factor-alpha and interleukin-1ß stimulated in the OGD-treated Neuro-2a (N-2a) cells. The levels of reactive oxygen species and malondialdehyde were enhanced while the superoxide dismutase levels were reduced by oxygen and glucose deprivation (OGD) treatment, in which the lincRNA-EPS overexpression could reverse this effect in the cells. LincRNA-EPS interacted with high-temperature requirement protein A1 (Htra1) and heterogeneous nuclear ribonucleoprotein L (HNRNPL), and their depletion inhibited the Htra1 mRNA stability in N-2a cells. HNRNPL knockdown blocked lincRNA-EPS overexpression-induced Htra1 expression in the cells. The depletion of Htra1 could rescue lincRNA-EPS overexpression-mediated N-2a cell injury, inflammation, and oxidative stress induced by OGD. Functionally, lincRNA-EPS alleviates CIR injury of the middle cerebral artery occlusion/reperfusion mice in vivo. In conclusion, lincRNA-EPS attenuates CIR injury by maintaining Htra1 stability through recruiting HNRNPL.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.