Initiation of hnRNPA1 Low-Complexity Domain Condensation Monitored by Dynamic Light Scattering.

Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating pH and salt conditions to perturb macromolecule and condensate properties. We observed the formation of nanometer-sized BMCs (nano-condensates) distinct from protein monomers and micron-sized condensates. We also observed that conditions that solubilize micron-sized protein condensates do not solubilize nano-condensates, indicating that the balance of forces that stabilize nano-condensates and micron-sized condensates are distinct. These findings provide insight into the forces that drive protein phase separation and potential nucleation structures of macromolecular condensation.

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

Multi-step regulation of microRNA expression and secretion into small extracellular vesicles by insulin.

Tissues release microRNAs (miRNAs) in small extracellular vesicles (sEVs) including exosomes, which can regulate gene expression in distal cells, thus acting as modulators of local and systemic metabolism. Here, we show that insulin regulates miRNA secretion into sEVs from 3T3-L1 adipocytes and that this process is differentially regulated from cellular expression. Thus, of the 53 miRNAs upregulated and 66 miRNAs downregulated by insulin in 3T3-L1 sEVs, only 12 were regulated in parallel in cells. Insulin regulated this process in part by phosphorylating hnRNPA1, causing it to bind to AU-rich motifs in miRNAs, mediating their secretion into sEVs. Importantly, 43% of insulin-regulated sEV-miRNAs are implicated in obesity and insulin resistance. These include let-7 and miR-103, which we show regulate insulin signaling in AML12 hepatocytes. Together, these findings demonstrate an important layer to insulin's regulation of adipose biology and provide a mechanism of tissue crosstalk in obesity and other hyperinsulinemic states.

Targeting Ser78 phosphorylation of Hsp27 achieves potent antiviral effects against enterovirus A71 infection.

A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27S15A and Hsp27S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2Apro) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27S82A. However, Hsp27S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

Downregulation of HNRNPA1 induced neoantigen generation via regulating alternative splicing.

BACKGROUND: Immunotherapies effectively treat human malignancies, but the low response and resistance are major obstacles. Neoantigen is an emerging target for tumor immunotherapy that can enhance anti-tumor immunity and improve immunotherapy. Aberrant alternative splicing is an important source of neoantigens. HNRNPA1, an RNA splicing factor, was found to be upregulated in the majority of tumors and play an important role in the tumor immunosuppressive microenvironment. METHODS: Whole transcriptome sequencing was performed on shHNRNPA1 SKOV3 cells and transcriptomic data of shHNRNPA1 HepG2, MCF-7M, K562, and B-LL cells were downloaded from the GEO database. Enrichment analysis was performed to elucidate the mechanisms underlying the activation of anti-tumor immunity induced by HNRNPA1 knockdown. mRNA alternative splicing was analyzed and neoantigens were predicted by JCAST v.0.3.5 and Immune epitope database. The immunogenicity of candidate neoantigens was calculated by Class I pMHC Immunogenicity and validated by the IFN-γ ELISpot assay. The effect of shHNRNPA1 on tumor growth and immune cells in vivo was evaluated by xenograft model combined with immunohistochemistry. RESULTS: HNRNPA1 was upregulated in a majority of malignancies and correlated with immunosuppressive status of the tumor immune microenvironment. Downregulation of HNRNPA1 could induce the activation of immune-related pathways and biological processes. Disruption of HNRNPA1 resulted in aberrant alternative splicing events and generation of immunogenic neoantigens. Downregulation of HNRNPA1 inhibited tumor growth and increased CD8+ T cell infiltration in vivo. CONCLUSION: Our study demonstrated that targeting HNRNPA1 could produce immunogenic neoantigens that elicit anti-tumor immunity by inducing abnormal mRNA splicing. It suggests that HNRNPA1 may be a potential target for immunotherapy.

SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13.

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion, and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion, and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer exists at the 3' splice site and 5' splice site of LOXL2 exon 13. HnRNPA1 can bind to the exonic splicing silencer and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

H3.3-G34W in giant cell tumor of bone functionally aligns with the exon choice repressor hnRNPA1L2.

RNA processing is an essential post-transcriptional phenomenon that provides the necessary complexity of transcript diversity prior to translation. Aberrations in this process could contribute to tumourigenesis, and we have previously reported increased splicing alterations in giant cell tumor of bone (GCTB), which carries mutations in the histone variant H3.3 encoding glycine 34 substituted for tryptophan (H3.3-G34W). G34W interacts with several splicing factors, most notably the trans-acting splicing factor hnRNPA1L2. To gain a deeper understanding of RNA processing in GCTB and isogenic HeLa cells with H3.3-G34W, we generated RNA-immunoprecipitation sequencing data from hnRNPA1L2 and H3.3-G34W associated RNAs, which showed that 80% overlapped across genic regions and were frequently annotated as E2F transcription factor binding sites. Splicing aberrations in both GCTB and HeLa cells with H3.3-G34W were significantly enriched for known hnRNPA1L2 binding motifs (p value < 0.01). This splicing aberration differed from hnRNPA1L2 knockouts, which showed alterations independent of H3.3-G34W. Of functional significance, hnRNPA1L2 was redistributed to closely match the H3.3 pattern, likely driven by G34W, and to loci not occupied in normal parental cells. Taken together, our data reveal a functional overlap between hnRNPA1L2 and H3.3-G34W with likely significant consequences for RNA processing during GCTB pathogenesis. This provides novel opportunities for therapeutic intervention in future modus operandi.

Genotype-phenotype association and functional analysis of hnRNPA1 mutations in amyotrophic lateral sclerosis.

BACKGROUND: Pathogenic variants in hnRNPA1 have been reported in amyotrophic lateral sclerosis (ALS) patients. However, studies on hnRNPA1 mutant spectrum and pathogenicity of variants were rare. METHODS: We performed whole exome sequencing of ALS-associated genes and subsequent verification of rare variants in hnRNPA1 in our ALS patients. The hnRNPA1 mutations reported in literature were reviewed and combined with our results to determine the genotype-phenotype relationship. Functional analysis of the novel variant p.G195A was performed in vitro by transfection of mutant hnRNPA1 into 293T cell. RESULTS: Among 207 ALS patients recruited, 3 rare hnRNPA1 variants were identified (mutant frequency 1.45%), including two recurrent mutations (p.P340S and p.G283R), and a novel rare variant p.G195A. In combination with previous reports, there are 27 ALS patients with 15 hnRNPA1 mutations identified. Disease onset age was 47.90 ± 1.52 years with predominant limb onset. The p.P340S mutation caused flail arm syndrome (FAS) in two independent families with extended life expectancy. The newly identified p.G195A mutation, lying at the start of the PrLD ("prion-like" domain)/LCD (low-complexity domain), causes local structural changes in 3D protein prediction. Upon sodium arsenite exposure, mutant hnRNPA1 retained in the nucleus but deficit of cytoplasmic G3BP1-positive stress granule clearance was observed. This is different from the p.P340S mutation which caused both cytoplasmic translocation and stress granule formation. No cytoplasmic TDP-43 translocation was observed. CONCLUSION: Mutations in hnRNPA1 are overall minor in ALS patients. The p.P340S mutation is associated with manifestation of FAS. Mutations in LCD of hnRNPA1 cause stress granule misprocessing.

circLIFR-007 reduces liver metastasis via promoting hnRNPA1 nuclear export and YAP phosphorylation in breast cancer.

Cancer metastasis is the major cause of death in patients with breast cancer (BC). The liver is a common site of breast cancer metastasis, and the 5-year survival rate of patients with breast cancer liver metastases (BCLMs) is only about 8.5 %. CircRNAs are involved in a variety of cancer-related pathological behaviors, and their unique structure and resistance to RNA degradation enable them to serve as ideal diagnostic biomarkers and therapeutic targets. Therefore, it is important to investigate the role and molecular mechanism of circRNAs in cancer metastasis. CircLIFR-007 was identified as a critical circular RNA in BC metastasis by circRNAs microarray and qRT-PCR experiment. Cell function assays were performed to explore the effect of circLIFR-007 in breast cancer cells. Experiments in vivo validated the function of circLIFR-007. Several molecular assays were performed to investigate the underlying mechanisms. We found that circLIFR-007 acted as a negative controller in breast cancer liver metastasis. CircLIFR-007 upregulates the phosphorylation level of YAP by exporting hnRNPA1 to promote the combination between hnRNPA1 and YAP in the cytoplasm. Overexpression of circLIFR-007 suppressed the expression of liver metastasis-related proteins, SREBF1 and SNAI1, which were regulated by transcription factor YAP. Functionally, circLIFR-007 inhibits the proliferation and metastasis of breast cancer cells both in vivo and in vitro.

RNA modulates hnRNPA1A amyloid formation mediated by biomolecular condensates.

Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein-RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid-protein interactions affect the kinetics and molecular pathways of amyloid formation.

Embryonic stem cell related gene regulates alternative splicing of transcription factor 3 to maintain human embryonic stem cells' self-renewal and pluripotency.

Exploring the mechanism of self-renewal and pluripotency maintenance of human embryonic stem cells (hESCs) is of great significance in basic research and clinical applications, but it has not been fully elucidated. Long non-coding RNAs (lncRNAs) have been shown to play a key role in the self-renewal and pluripotency maintenance of hESCs. We previously reported that the lncRNA ESRG, which is highly expressed in undifferentiated hESCs, can maintain the self-renewal and pluripotency of hPSCs. RNA pull-down mass spectrometry showed that ESRG could bind to other proteins, among which heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) attracted our attention. In this study, we showed that HNRNPA1 can maintain self-renewal and pluripotency of hESCs. ESRG bound to and stabilized HNRNPA1 protein through the ubiquitin-proteasome pathway. In addition, knockdown of ESRG or HNRNPA1 resulted in alternative splicing of TCF3, which originally and primarily encoded E12, to mainly encode E47 and inhibit CDH1 expression. HNRNPA1 could rescue the biological function changes of hESCs caused by ESRG knockdown or overexpression. Our results suggest that ESRG regulates the alternative splicing of TCF3 to affect CDH1 expression and maintain hESCs self-renewal and pluripotency by binding and stabilizing HNRNPA1 protein. This study lays a good foundation for exploring the new molecular regulatory mechanism by which ESRG maintains hESCs self-renewal and pluripotency.

m6A-Induced lncRNA MEG3 Promotes Cerebral Ischemia-Reperfusion Injury Via Modulating Oxidative Stress and Mitochondrial Dysfunction by hnRNPA1/Sirt2 Axis.

Ischemic stroke remains one of the major causes of serious disability and death globally. LncRNA maternally expressed gene 3 (MEG3) is elevated in middle cerebral artery occlusion/reperfusion (MCAO/R) rats and oxygen-glucose deprivation/reperfusion (OGD/R)-treated neurocytes cells. The objective of this study is to investigate the mechanism underlying MEG3-regulated cerebral ischemia/reperfusion (I/R) injury. MCAO/R mouse model and OGD/R-treated HT-22 cell model were established. The cerebral I/R injury was monitored by TTC staining, neurological scoring, H&E and TUNEL assay. The levels of MEG3, hnRNPA1, Sirt2 and other key molecules were detected by qRT-PCR and western blot. Mitochondrial dysfunction was assessed by transmission Electron Microscopy (TEM), JC-1 and MitoTracker staining. Oxidative stress was monitored using commercial kits. Bioinformatics analysis, RIP, RNA pull-down assays and RNA FISH were employed to detect the interactions among MEG3, hnRNPA1 and Sirt2. The m6A modification of MEG3 was assessed by MeRIP-qPCR. MEG3 promoted MCAO/R-induced brain injury by modulating mitochondrial fragmentation and oxidative stress. It also facilitated OGD/R-induced apoptosis, mitochondrial dysfunction and oxidative stress in HT-22 cells. Mechanistically, direct associations between MEG3 and hnRNPA1, as well as between hnRNPA1 and Sirt2, were observed in HT-22 cells. MEG3 regulated Sirt2 expression in a hnRNPA1-dependent manner. Functional studies showed that MEG3/Sirt2 axis contributed to OGD/R-induced mitochondrial dysfunction and oxidative stress in HT-22 cells. Additionally, METTL3 was identified as the m6A transferase responsible for the m6A modification of MEG3. m6A-induced lncRNA MEG3 promoted cerebral I/R injury via modulating oxidative stress and mitochondrial dysfunction by hnRNPA1/Sirt2 axis.

Adipocyte-Specific Hnrnpa1 Knockout Aggravates Obesity-Induced Metabolic Dysfunction via Upregulation of CCL2.

Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) is involved in lipid and glucose metabolism via mRNA processing. However, whether and how HNRNPA1 alters adipocyte function in obesity remain obscure. Here, we found that the obese state downregulated HNRNPA1 expression in white adipose tissue (WAT). The depletion of adipocyte HNRNPA1 promoted markedly increased macrophage infiltration and expression of proinflammatory and fibrosis genes in WAT of obese mice, eventually leading to exacerbated insulin sensitivity, glucose tolerance, and hepatic steatosis. Mechanistically, HNRNPA1 interacted with Ccl2 and regulated its mRNA stability. Intraperitoneal injection of CCL2-CCR2 signaling antagonist improved adipose tissue inflammation and systemic glucose homeostasis. Furthermore, HNRNPA1 expression in human WAT was negatively correlated with BMI, fat percentage, and subcutaneous fat area. Among individuals with 1-year metabolic surgery follow-up, HNRNPA1 expression was positively related to percentage of total weight loss. These findings identify adipocyte HNRNPA1 as a link between adipose tissue inflammation and systemic metabolic homeostasis, which might be a promising therapeutic target for obesity-related disorders.

Phosphorylation of hnRNP A1-Serine 199 Is Not Required for T Cell Differentiation and Function.

hnRNP A1 is an important RNA-binding protein that influences many stages of RNA processing, including transcription, alternative splicing, mRNA nuclear export, and RNA stability. However, the role of hnRNP A1 in immune cells, specifically CD4+ T cells, remains unclear. We previously showed that Akt phosphorylation of hnRNP A1 was dependent on TCR signal strength and was associated with Treg differentiation. To explore the impact of hnRNP A1 phosphorylation by Akt on CD4+ T cell differentiation, our laboratory generated a mutant mouse model, hnRNP A1-S199A (A1-MUT) in which the major Akt phosphorylation site on hnRNP A1 was mutated to alanine using CRISPR Cas9 technology. Immune profiling of A1-MUT mice revealed changes in the numbers of Tregs in the mesenteric lymph node. We found no significant differences in naive CD4+ T cell differentiation into Th1, Th2, Th17, or T regulatory cells (Tregs) in vitro. In vivo, Treg differentiation assays using OTII-A1-Mut CD4+ T cells exposed to OVA food revealed migration and homing defects in the A1-MUT but no change in Treg induction. A1-MUT mice were immunized with NP- keyhole limpet hemocyanin, and normal germinal center development, normal numbers of NP-specific B cells, and no change in Tfh numbers were observed. In conclusion, Akt phosphorylation of hnRNP A1 S199 does not play a role in CD4+ T cell fate or function in the models tested. This hnRNP A1-S199A mouse model should be a valuable tool to study the role of Akt phosphorylation of hnRNP A1-S199 in different cell types or other mouse models of human disease.

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.

hnRNP A1 dysfunction alters RNA splicing and drives neurodegeneration in multiple sclerosis (MS).

Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.

The microRNA-195-5p/hnRNP A1 axis contributes to the progression of hepatocellular carcinoma by regulating the migration of cancer cells.

Dysregulation of gene expression is critical for the progression of cancer. The augmented expression of hnRNP A1 in patients with hepatocellular carcinoma (HCC) has been related to its oncogenic functions. However, the underlying mechanisms responsible for upregulation of hnRNP A1 have not been fully elucidated. In the present study, we identified microRNA-195-5p (miR-195-5p), a miRNA downregulated in HCC, as a novel regulator governing hnRNP A1 expression. Notably, our investigations showed an inverse correlation between hnRNP A1 level, which was increased in HCC, and miR-195-5p level, which was decreased. Our findings demonstrated that hnRNP A1 significantly enhanced the migration and invasion of PLC/PRF/5 cells through its association with mRNAs regulating metastasis. MiR-195-5p also interfered with the hnRNP A1-mediated cell migration by targeting hnRNP A1. Our results underscore the significance of the miR-195-5p/hnRNP A1 axis in regulating the migratory potential of cancer cells and its role in promoting HCC by orchestrating cell migration processes.

M2 Macrophage-Derived Exosomal lncRNA MIR4435-2HG Promotes Progression of Infantile Hemangiomas by Targeting HNRNPA1.

Purpose: Infantile hemangiomas (IHs) are commonly observed benign tumors that can cause serious complications. M2-polarized macrophages in IHs promote disease progression. In this study, we investigated the role of M2 macrophage-derived exosomal lncRNA MIR4435-2HG in IHs. Patients and Methods: Exosomes derived from M2 polarized macrophages were extracted. Next, using cell co-culture or transfection, we investigated whether M2 polarized macrophage-derived exosomes (M2-exos) can transport MIR4435-2HG to regulate the proliferation, migration, invasion, and angiogenesis of hemangioma-derived endothelial cells (HemECs). RNA-seq and RNA pull-down assays were performed to identify targets and regulatory pathways of MIR4435-2HG. We explored the possible mechanisms through which MIR4435-2HG regulates the biological function of HemECs. Results: M2-exos significantly enhanced the proliferation, migration, invasion, and angiogenesis of HemECs. Thus, HemECs uptake M2-exos and promote biological functions through the inclusion of MIR4435-2HG. RNA-seq and RNA pull-down experiments confirmed that MIR4435-2HG regulates of HNRNPA1 expression and directly binds to HNRNPA1, consequently affecting the NF-κB signal pathway. Conclusion: MIR4435-2HG of M2-exos promotes the progression of IHs and enhances the proliferation, migration, invasion, and angiogenesis of HemECs by directly binding to HNRNPA1. This study not only reveals the mechanism of interaction between M2 macrophages and HemECs, but also provides a promising therapeutic target for IHs.

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex.

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the "TAG" binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.