Exosomes secreted by Fusobacterium nucleatum-infected colon cancer cells transmit resistance to oxaliplatin and 5-FU by delivering hsa_circ_0004085.

BACKGROUND: A large number of Fusobacterium nucleatum (Fn) are present in colorectal cancer (CRC) tissues of patients who relapse after chemotherapy, and Fn has been reported to promote oxaliplatin and 5-FU chemoresistance in CRC. Pathogens such as bacteria and parasites stimulate exosome production in tumor cells, and the regulatory mechanism of exosomal circRNA in the transmission of oxaliplatin and 5-FU chemotherapy resistance in Fn-infected CRC remains unclear. METHODS: Hsa_circ_0004085 was screened by second-generation sequencing of CRC tissues. The correlation between hsa_circ_0004085 and patient clinical response to oxaliplatin/5-FU was analyzed. Exosome tracing experiments and live imaging systems were used to test the effect of Fn infection in CRC on the distribution of hsa_circ_0004085. Colony formation, ER tracking analysis and immunofluorescence were carried out to verify the regulatory effect of exosomes produced by Fn-infected CRC cells on chemotherapeutic resistance and ER stress. RNA pulldown, LC-MS/MS analysis and RIP were used to explore the regulatory mechanism of downstream target genes by hsa_circ_0004085. RESULTS: First, we screened out hsa_circ_0004085 with abnormally high expression in CRC clinical samples infected with Fn and found that patients with high expression of hsa_circ_0004085 in plasma had a poor clinical response to oxaliplatin/5-FU. Subsequently, the circular structure of hsa_circ_0004085 was identified. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes produced by Fn-infected CRC cells transferred hsa_circ_0004085 between cells and delivered oxaliplatin/5-FU resistance to recipient cells by relieving ER stress. Hsa_circ_0004085 enhanced the stability of GRP78 mRNA by binding to RRBP1 and promoted the nuclear translocation of ATF6p50 to relieve ER stress. CONCLUSIONS: Plasma levels of hsa_circ_0004085 are increased in colon cancer patients with intracellular Fn and are associated with a poor response to oxaliplatin/5-FU. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes secreted by Fn-infected CRC cells deliver hsa_circ_0004085 between cells. Hsa_circ_0004085 relieves ER stress in recipient cells by regulating GRP78 and ATF6p50, thereby delivering resistance to oxaliplatin and 5-FU.

Lnc-PPP2R1B Mediates the Alternative Splicing of PPP2R1B by Interacting and Stabilizing HNRNPLL and Promotes Osteogenesis of MSCs.

Osteogeinc differentiation from mesenchymal stem cells (MSCs) into osteoblasts is a key step for bone tissue engineering in regenerative medicine. The insight into regulatory mechanism of osteogenesis of MSCs facilitates achieving better recovery effect. Long non-coding RNAs are regarded as a family of important moderators in osteogenesis. In this study, we found a novel lncRNA, lnc-PPP2R1B was up-regulated during osteogenesis of MSCs by Illumina HiSeq transcritome sequencing. We demonstrated lnc-PPP2R1B overexpression promoted osteogenesis and knockdown of lnc-PPP2R1B inhibited osteogenesis of MSCs. Mechanically, it physically interacted with and up-regulated heterogeneous nuclear ribonucleoprotein L Like (HNRNPLL), which is a master regulator of activation-induced alternative splicing in T cells. We found lnc-PPP2R1B knockdown or HNRNPLL knockdown decreased transcript-201 of Protein Phosphatase 2A, Regulatory Subunit A, Beta Isoform (PPP2R1B) while increased transcript-203 of PPP2R1B, and did not affect transcript-202/204/206. PPP2R1B is a constant regulatory subunit of protein phosphatase 2 (PP2A), which activates Wnt/ß-catenin pathway by removing phosphorylation and stabilization of ß-catenin and translocation into nucleus. The transcript-201 retained exon 2 and 3, compared to transcript-203. And it was reported the exon 2 and 3 of PPP2R1B were one part of B subunit binding domain on A subunit in PP2A trimer, and therefore retaining exon 2 and 3 promised formation and enzyme function of PP2A. Finally, lnc-PPP2R1B promoted ectopic osteogenesis in vivo. Conclusively, lnc-PPP2R1B mediated alternative splicing of PPP2R1B through retaining exon 2 and 3 by interacting with HNRNPLL and then promoted osteogenesis, which may facilitate an in-depth understanding of function and mechanism of lncRNAs in osteogenesis. Lnc-PPP2R1B interacted with HNRNPLL, and regulated alternative splicing of PPP2R1B through retaining exon 2 and 3, which preserved enzyme function of PP2A and enhanced dephosphorylation and nuclear translocation of ß-catenin, thereby promoting Runx2 and OSX expression and then osteogenesis. And it provided experimental data and potential target for promoting bone formation and bone regeneration.

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.

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.

HNRNPL induced circFAM13B increased bladder cancer immunotherapy sensitivity via inhibiting glycolysis through IGF2BP1/PKM2 pathway.

BACKGROUND: The response rate to immunotherapy in patients with bladder cancer (BCa) remains relatively low. Considering the stable existence and important functions in tumour metabolism, the role of circRNAs in regulating immune escape and immunotherapy sensitivity is receiving increasing attention. METHODS: Circular RNA (circRNA) sequencing was performed on five pairs of BCa samples, and circFAM13B (hsa_circ_0001535) was screened out because of its remarkably low expression in BCa. Further mRNA sequencing was conducted, and the association of circFAM13B with glycolysis process and CD8+ T cell activation was confirmed. The functions of circFAM13B were verified by proliferation assays, glycolysis assays, BCa cells-CD8+ T cell co-culture assays and tumorigenesis experiment among human immune reconstitution NOG mice. Bioinformatic analysis, RNA-protein pull down, mass spectrometry, RNA immunoprecipitation, luciferase reporter assay and fluorescence in situ hybridization were performed to validate the HNRNPL/circFAM13B/IGF2BP1/PKM2 cascade. RESULTS: Low expression of circFAM13B was observed in BCa, and it was positively associated with lower tumour stage and better prognosis among patients with BCa. The function of CD8+ T cells was promoted by circFAM13B, and it could attenuate the glycolysis of BCa cells and reverse the acidic tumour microenvironment (TME). The production of granzyme B and IFN-γ was improved, and the immunotherapy (PD-1 antibodies) sensitivity was facilitated by the inhibition of acidic TME. Mechanistically, circFAM13B was competitively bound to the KH3-4 domains of IGF2BP1 and subsequently reduced the binding of IGF2BP1 and PKM2 3'UTR. Thus, the stability of the PKM2 mRNA decreased, and glycolysis-induced acidic TME was inhibited. The generation of circFAM13B was explored by confirming whether heterogeneous nuclear ribonucleoprotein L (HNRNPL) could promote circFAM13B formation via pre-mRNA back-splicing. CONCLUSIONS: HNRNPL-induced circFAM13B could repress immune evasion and enhance immunotherapy sensitivity by inhibiting glycolysis and acidic TME in BCa through the novel circFAM13B/IGF2BP1/PKM2 cascade. Therefore, circFAM13B can be used as a biomarker for guiding the immunotherapy among patients with BCa.

Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

MOBT Alleviates Pulmonary Fibrosis through an lncITPF-hnRNP-l-Complex-Mediated Signaling Pathway.

Pulmonary fibrosis is characterized by the destruction of alveolar architecture and the irreversible scarring of lung parenchyma, with few therapeutic options and effective therapeutic drugs. Here, we demonstrate the anti-pulmonary fibrosis of 3-(4-methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl(αS)-α,3,4-trihydroxybenzenepropanoate (MOBT) in mice and a cell model induced by bleomycin and transforming growth factor-ß1. The anti-pulmonary fibrosis of MOBT was evaluated using a MicroCT imaging system for small animals, lung function analysis and H&E and Masson staining. The results of RNA fluorescence in situ hybridization, chromatin immunoprecipitation (ChIP)-PCR, RNA immunoprecipitation, ChIP-seq, RNA-seq, and half-life experiments demonstrated the anti-pulmonary fibrotic mechanism. Mechanistic dissection showed that MOBT inhibited lncITPF transcription by preventing p-Smad2/3 translocation from the cytoplasm to the nucleus, resulting in a reduction in the amount of the lncITPF-hnRNP L complex. The decreased lncITPF-hnRNP L complex reduced MEF2c expression by blocking its alternative splicing, which in turn inhibited the expression of MEF2c target genes, such as TAGLN2 and FMN1. Briefly, MOBT alleviated pulmonary fibrosis through the lncITPF-hnRNP-l-complex-targeted MEF2c signaling pathway. We hope that this study will provide not only a new drug candidate but also a novel therapeutic drug target, which will bring new treatment strategies for pulmonary fibrosis.

Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1.

Alternative splicing plays key roles for cell type-specific regulation of protein function. It is controlled by cis-regulatory RNA elements that are recognized by RNA binding proteins (RBPs). The MALT1 paracaspase is a key factor of signaling pathways that mediate innate and adaptive immune responses. Alternative splicing of MALT1 is critical for controlling optimal T cell activation. We demonstrate that MALT1 splicing depends on RNA structural elements that sequester the splice sites of the alternatively spliced exon7. The RBPs hnRNP U and hnRNP L bind competitively to stem-loop RNA structures that involve the 5' and 3' splice sites flanking exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L disrupts these RNA elements to facilitate recruitment of the essential splicing factor U2AF2, thereby promoting exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.

The m6A-induced lncRNA CASC8 promotes proliferation and chemoresistance via upregulation of hnRNPL in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) are dysregulated in many cancers. Here, we identified the molecular mechanisms of lncRNA Cancer Susceptibility Candidate 8 (CASC8) in promoting the malignancy of esophageal squamous cell carcinoma (ESCC). CASC8 was highly overexpressed in ESCC tissues and upregulation of CASC8 predicted poor prognosis in ESCC patients. Moreover, CASC8 decreased the cisplatin sensitivity of ESCC cells and promoted ESCC tumor growth in vivo. Mechanistically, CASC8 interacted with heterogeneous nuclear ribonucleoprotein L (hnRNPL) and inhibited its polyubiquitination and proteasomal degradation, thus stabilizing hnRNPL protein levels and activating the Bcl2/caspase3 pathway. Additionally, AlkB Homolog 5, RNA demethylase (ALKBH5)-mediated m6A demethylation stabilized the CASC8 transcript, resulting in CASC8 upregulation. Taken together, these findings identified an oncogenic function of CASC8 in the progression of ESCC, which suggest that CASC8 might become a potential prognostic biomarker in ESCC.

Cortex metabolome and proteome analysis reveals chronic arsenic exposure via drinking water induces developmental neurotoxicity through hnRNP L mediated mitochondrial dysfunction in male rats.

Lots of people are at the risk of arsenic-contaminated drinking water. Arsenic exposure was confirmed to be closely linked to neurocognitive deficits, particularly during childhood. The multi-omics approaches are known be well suitable for toxicological research. Thus, this study aimed to explore the molecular mechanisms of arsenic-induced learning and memory function impairments through the integrative proteome and metabolome analysis of cortex in rats. The weaned rats were exposed to arsenic-contaminated drinking water for six months to mimic the developmental exposure. 220 differential proteins and 19 differential metabolites were identified in the cortex, and nine potential biomarkers were found to be related to impaired Morris water maze (MWM) indicators. Chronic arsenic exposure affected the cognitive function by inducing the overproduction of amyloid-ß (Aß) peptides and the redox imbalance in the mitochondria. Glycolysis and tricarboxylic acid (TCA) cycle enhancement driven by the increased heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a low-dose protective mechanism against arsenic-induced ATP deficiency and oxidative stress. Moreover, apoptosis is another important pathway of arsenic-induced neurotoxicity. This study provides new evidence about the alterations of proteins and metabolites in the cortex of the exposed rats under arsenic toxicity. These findings suggest hnRNP L could be a potential target for the treatment of arsenic-induced neurotoxicity.

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.

Regulation of integrin and extracellular matrix genes by HNRNPL is necessary for epidermal renewal.

Stratified epithelia such as the epidermis require coordinated regulation of stem and progenitor cell proliferation, survival, and differentiation to maintain homeostasis. Integrin-mediated anchorage of the basal layer stem cells of the epidermis to the underlying dermis through extracellular matrix (ECM) proteins is crucial for this process. It is currently unknown how the expression of these integrins and ECM genes are regulated. Here, we show that the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to these genes on chromatin to promote their expression. HNRNPL recruits RNA polymerase II (Pol II) to integrin/ECM genes and is required for stabilizing Pol II transcription through those genes. In the absence of HNRNPL, the basal layer of the epidermis where the stem cells reside prematurely differentiates and detaches from the underlying dermis due to diminished integrin/ECM expression. Our results demonstrate a critical role for RBPs on chromatin to maintain stem and progenitor cell fate by dictating the expression of specific classes of genes.

FBXO16-mediated hnRNPL ubiquitination and degradation plays a tumor suppressor role in ovarian cancer.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a type of RNA binding protein that highly expressed in a variety of tumors and plays a vital role in tumor progression. However, its post-translational regulation through ubiquitin-mediated proteolysis and the cellular mechanism responsible for its proteasomal degradation remains unclear. F-box proteins (FBPs) function as the substrate recognition subunits of SCF ubiquitin ligase complexes and directly bind to substrates. The aberrant expression or mutation of FBPs will lead to the accumulation of its substrate proteins that often involved in tumorigenesis. Here we discover FBXO16, an E3 ubiquitin ligase, to be a tumor suppressor in ovarian cancer, and patients with the relatively high expression level of FBXO16 have a better prognosis. Silencing or depleting FBXO16 significantly enhanced ovarian cancer cell proliferation, clonogenic survival, and cell invasion by activating multiple oncogenic pathways. This function requires the F-box domain of FBXO16, through which FBXO16 assembles a canonical SCF ubiquitin ligase complex that constitutively targets hnRNPL for degradation. Depletion of hnRNPL is sufficient to inactive multiple oncogenic signaling regulated by FBXO16 and prevent the malignant behavior of ovarian cancer cells caused by FBXO16 deficiency. FBXO16 interacted with the RRM3 domain of hnRNPL via its C-terminal region to trigger the proteasomal degradation of hnRNPL. Failure to degrade hnRNPL promoted ovarian cancer cell proliferation in vitro and tumor growth vivo, phenocopying the deficiency of FBXO16 in ovarian cancer.

The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain.

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

The MTNR1A mRNA is stabilized by the cytoplasmic hnRNPL in renal tubular cells.

The downregulation of melatonin receptor 1A (MTNR1A) is associated with a range of pathological conditions, including membranous nephropathy. Knowledge of the mechanism underlying MTNR1A expression has been limited to the transcriptional regulation level. Here, RNA interference screening in human kidney cells revealed that heterogeneous nuclear ribonucleoprotein L (hnRNPL) upregulated MTNR1A RNA post-transcriptionally. hnRNPL knockdown or overexpression led to increased or decreased levels of cyclic adenosine monophosphate-responsive element-binding protein phosphorylation, respectively. Molecular studies showed that cytoplasmic hnRNPL exerts a stabilizing effect on the MTNR1A transcript through CA-repeat elements in its coding region. Further studies revealed that the interaction between hnRNPL and MTNR1A serves to protect MNTR1A RNA degradation by the exosome component 10 protein. MTNR1A, but not hnRNPL, displays a diurnal rhythm in mouse kidneys. Enhanced levels of MTNR1A recorded at midnight correlated with robust binding activity between cytoplasmic hnRNPL and the MTNR1A transcript. Both hnRNPL and MTNR1A were decreased in the cytoplasm of tubular epithelial cells from experimental membranous nephropathy kidneys, supporting their clinical relevance. Collectively, our data identified cytoplasmic hnRNPL as a novel player in the upregulation of MTNR1A expression in renal tubular epithelial cells, and as a potential therapeutic target.

Design and application of circular RNAs with protein-sponge function.

Circular RNAs (circRNAs) are a class of noncoding RNAs, generated from pre-mRNAs by circular splicing of exons and functionally largely uncharacterized. Here we report on the design, expression, and characterization of artificial circRNAs that act as protein sponges, specifically binding and functionally inactivating hnRNP (heterogeneous nuclear ribonucleoprotein) L. HnRNP L regulates alternative splicing, depending on short CA-rich RNA elements. We demonstrate that designer hnRNP L-sponge circRNAs with CA-repeat or CA-rich sequence clusters can efficiently and specifically modulate splicing-regulatory networks in mammalian cells, including alternative splicing patterns and the cellular distribution of a splicing factor. This new strategy can in principle be applied to any RNA-binding protein, opening up new therapeutic strategies in molecular medicine.

Roles of heterogeneous nuclear ribonucleoprotein L in enamel organ development and the differentiation of ameloblasts.

OBJECTIVE: We aimed to explore the role of Heterogeneous Nuclear Ribonucleoprotein L(hnRNP L) in enamel organ development through hnRNP L conditional knockout mice and knockdown of hnRNP L expression in mouse ameloblast-lineage cells (mALCs) METHODS: We created K14cre-mediated hnRNP L conditional knockout mice (hnRNP LK14/fl) and silenced the expression of hnRNP L in mALCs to investigate the role of hnRNP L in enamel organ development. RESULTS: We found that hnRNP LK14/fl mice presented enamel organ development defects with reduced number of inner enamel epithelium (IEE) cells. The proliferation and differentiation of the IEE cells/ameloblasts were suppressed. The cell proliferation and mineralization ability were also decreased after hnRNP L knockdown. Further studies showed that Bone Morphogenetic Protein (BMP) signaling pathway was attenuated after the knockdown of hnRNP L expression both in vivo and in vitro. CONCLUSIONS: These findings suggest that hnRNP L plays a critical role in enamel organ development by promoting the IEE cell/ameloblast proliferation and differentiation. BMP signaling pathway may be involved in the process.

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.

Heterogeneous Nuclear Ribonucleoprotein L Negatively Regulates Foot-and-Mouth Disease Virus Replication through Inhibition of Viral RNA Synthesis by Interacting with the Internal Ribosome Entry Site in the 5' Untranslated Region.

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3Dpol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3Dpol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.