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Flail arm syndrome (FAS) is one of the atypical subtypes of amyotrophic lateral sclerosis (ALS). Mutations in hnRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare genetic cause of ALS. Herein, marked clinical heterogeneity of FAS in a pedigree with a known hnRNPA1 variant was described to raise early awareness of the ALS variant. Furtherly, a literature review of the hnRNPA1-related spectrum was made to summarize the clinical and genetic characteristics.Detailed clinical evaluation, muscle pathology, and whole-exome sequencing were performed. The sequence and co-segregation of the mutation among the family members were confirmed by Sanger sequencing.The great clinical variability was found in a FAS pedigree. Muscle pathology revealed a cluster distribution of angulated or rounded atrophic fibers, accompanied by significant multi-nucleus aggregation. Immunohistochemical staining showed that mutant hnRNPA1 proteins accumulated in muscle fiber cytoplasm. Exome sequencing identified a documented variant in hnRNPA1 gene c.1018C T (p.P340S), which co-segregated with disease in the family. Besides, highly phenotypic heterogeneity was also found in other hnRNPA1-related diseases.We described a Chinese pedigree with hnRNPA1-related FAS, which showed significant clinical variability among the intrafamilial members. FAS is a relatively milder variant of ALS, due to the highly heterogeneous clinical spectrum, early observation is of paramount importance. In addition, the highly phenotypic heterogeneity and molecular genetic mechanism of the hnRNPA1-related spectrum are still beyond fully understood. Further, the detailed molecular mechanism underlying the clinical diversity is warranted to be explored.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are essential players in the regulation of gene expression. The majority of the twenty different hnRNP proteins act through the modulation of pre-mRNA splicing. Most have been shown to regulate the expression of critical genes for the progression of tumorigenic processes and were also observed to be overexpressed in several types of cancer. Moreover, these proteins were described as essential components for the maturation of some microRNAs (miRNAs). In the human genome, over 70% of miRNAs are transcribed from introns; therefore, we hypothesized that regulatory proteins involved with splicing could be important for their maturation. Increased expression of the miR-17-92 cluster has already been shown to be related to the development of many cancers, such as thyroid, lung, and lymphoma. In this article, we show that overexpression of hnRNP A1 and hnRNP C in BCPAP thyroid cancer cells directly affects the expression of miR-17-92 miRNAs. Both proteins associate with the 5'-end of this cluster, strongly precipitate miRNAs miR-17 and miR-18a and upregulate the expression of miR-92a. Upon overexpression of these hnRNPs, BCPAP cells also show increased proliferation, migration, and invasion rates, suggesting upregulation of these proteins and miRNAs is related to an enhanced tumorigenic phenotype.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), telomeric repeat-containing RNA (TERRA), and protection of telomeres 1 (POT1) have been reported to orchestrate to displace replication protein A (RPA) from telomeric overhangs, ensuring orderly telomere replication and capping. Our previous studies further demonstrated that DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-dependent hnRNPA1 phosphorylation plays a crucial role in the promotion of hnRNPA1 binding to telomeric overhangs and RPA displacement during G2–M phases. However, it is unclear that how the subsequent exchange between hnRNPA1 and POT1 is orchestrated. Here we report that the protein phosphatase 2A (PP2A) depends on its scaffold subunit, which is called PPP2R1A, to interact with and dephosphorylate hnRNPA1 in the late M phase. Furthermore, PP2A-mediated hnRNPA1 dephosphorylation and TERRA accumulation act in concert to promote the hnRNPA1-to-POT1 switch on telomeric single-stranded DNA. Consequently, defective PPP2R1A results in ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage response at telomeres as well as induction of fragile telomeres. Combined inhibition of ATR and PP2A induces entry into a catastrophic mitosis and leads to synthetic lethality of tumor cells. In addition, PPP2R1A levels correlate with clinical stages and prognosis of multiple types of cancers. Taken together, our results indicate that PP2A is critical for telomere maintenance. Implications: This study demonstrates that the PP2A-dependent hnRNPA1 dephosphorylation and TERRA accumulation facilitates the formation of the protective capping structure of newly replicated telomeres, thus exerting essential oncogenic role in tumorigenesis.

Alzheimer's disease (AD) and frontotemporal dementia (FTD) overlap clinically and pathologically. However, the role of FTD-associated genes in patients with AD remained unclear. To explore the relationship between FTD-associated genes and AD risk, we investigated 14 FTD-associated genes via targeted next-generation sequencing panel or whole-genome sequencing in a total of 721 AD patients and 1391 controls. Common variant-based association analysis and gene-based association test of rare variants were performed by PLINK 1.9 and Sequence Kernel Association Test-Optimal (SKAT-O test) respectively. As a result, 2 common variants, UBQLN1 rs1044175 (p value = 2.76 × 10−4) and MAPT rs2258689 (p value = 5.71 × 10−4), differed significantly between AD patients and controls. Additionally, gene-based analysis aggregating rare variants demonstrated that HNRNPA1 reached statistical significance in the SKAT-O test (p value = 2.24 × 10−3). Protein–protein interaction analysis showed that UBQLN1, MAPT, and HNRNPA1 interacted with proteins encoded by well-recognized AD-associated genes. Our study indicated that UBQLN1, MAPT, and HNRNPA1 are implicated in the pathogenesis of AD in the mainland Chinese population.

The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)—coded for by a long noncoding RNA—whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1., Our study demonstrates that reticular stress in melanoma cells promoted the translocation of the IRES transacting factor hnRPN‐A1 to the cytoplasm. By binding to the IRES site of meloe mRNA, hnRPN‐A1 promoted the translation of the melanoma‐specific antigen MELOE‐1 and led to the expression of a new immunogenic HLA/peptide complex at the cell surface. Altogether, our data suggest that pharmacological stimulation of stress pathways may enhance the efficacy of T‐cell based immunotherapies in targeting stress‐induced tumor antigens, such as MELOE‐1 in patients with melanoma.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in the world. Although increasing evidence supports the role of heterogeneous ribonucleoprotein particle A1 (HNRNP A1) in tumor progression, the function of HNRNP A1 in HCC remains unclear. Here, we focused on the role of HNRNP A1 in the development of HCC. In this study, we found HNRNP A1 participates in many aspects of HCC, such as progression and prognosis. Our results showed that HNRNP A1 is upregulated in human HCC tissues and cell lines. High expression of HNRNP A1 can promote the proliferation, migration, and invasion in HCC cells and accelerate tumor progression in mice. Moreover, we found that HNRNP A1 prevents the senescence process of HCC cells. Knocking down of HNRNP A1 promotes the expression of P16INK4, which arrests the cell cycle and then induces the senescence phenotype in HCC cells. Furthermore, we found that HNRNP A1 regulated necroptosis and mitochondrial dynamics. In summary, our study indicates that HNRNP A1 promotes the development of HCC, which suggests a potential therapeutic target for HCC.

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.

Regulatory T cells (Tregs) are indispensable for the maintenance of immunological self-tolerance and homeostasis. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is required for optimal Treg induction. Here, we reveal that human-induced Tregs (iTregs) lacking hnRNPA1 show reduced expression of the transcription factor FOXP3, increased ubiquitination level of FOXP3, and impaired suppressive abilities. Human naïve CD4 T cells with hnRNPA1 knockdown show a decreased Treg differentiation ratio. hnRNPA1 could interact with FOXP3 as well as with the E3 ligase Stub1. The phosphorylation at hnRNPA1 S199 could increase both interactions. The overexpression of FOXP3 in Tregs containing shhnRNPA1 could not recover the phenotype caused by hnRNPA1 knockdown. Therefore, there might be multiple essential pathways regulated by hnRNPA1 in Tregs. In conclusion, we present a new role of hnRNPA1 in promoting Treg function, indicating it as a promising target for tumor therapies.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in Egypt. HCCs usually have a poor prognosis because of late diagnosis, aggressive metastasis, and early invasion. Heterogeneous ribonucleoproteins (HnRNPs) are nuclear proteins that play a variety of roles in telomere formation, DNA repair, cell signaling, and gene regulation.Zincfinger Eboxbinding homeoboxes (ZEBs) are transcription factors that have a consistent inverse correlation with Ecadherin in numerous types of cancer and associated with poor prognosis.This study aimed to verify the prognostic expression of HnRNP A1, ZEB1, and E-cadherin in HCC.The retrospective study consisted of 54 formalin-fixed paraffin-embedded tissue blocks of hepatocellular carcinoma.Immunohistochemical staining was performed using antibodies against HnRNP A1, ZEB1, and E-cadherin. The patients were followed at the Clinical Oncology Department from May 2018 to July 2021.SPSS version 20 using the Chi-square test to compare data and the Kaplan-Meier plot for comparing survival.HnRNP A1 high positivity was detected in 59.3% of the cases, whereas negative E-cadherin and ZEB 1 expression presented in 37% and 70.4% of the patients, respectively. A statistically significant relation was present between HnRNP A1, ZEB1, E-cadherin, and various clinicopathological variables. The mean progression-free survival and overall survival in low HnRNP A1 and negative ZEB1 expressions were longer than those exhibited in high HnRNP A1 and positive ZEB1 expressions.HnRNP A1 and ZEB1 expressions are poor prognostic factors of HCC. E-cadherin has an important role in the development of differentiated HCCs and favorable outcome.

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms. Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was funded by the NTU Integrated Medical Biological and Environmental Life Sciences NIM/02/2017 (to X.R. and D.L.), Academic Research Fund Tier 1 (RG22/21) from Singapore’s Ministry of Education (to X.R.), NRF2019-NRF-ISF003-3104 from the National Research Foundation of Singapore (to X.R.), and the OFIRG17nov084 grant from Singapore’s National Medical Research Council (to D.L.).

NOTCH1 mutation is an essential molecular biologic aberration in chronic lymphocytic leukemia (CLL). CLL patients with NOTCH1 mutation have shown an unfavorable survival and a poor response to chemoimmunotherapy. This study aims to present the mechanisms of adverse prognosis caused by NOTCH1 mutation from the perspective of the splicing factor heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).The microarray data in Gene Expression Omnibus datasets were analyzed by bioinformatics and the function of hnRNPA1 was checked by testing the proliferation and apoptosis of CLL-like cell lines. Afterward, quantitative reverse transcription-polymerase chain reaction and Western blotting were applied to explore the relationship among NOTCH1, c-Myc, and hnRNPA1.RNA splicing was found to play a vital part in NOTCH1-mutated CLL cells; hence, hnRNPA1 was selected as the focus of this study. Higher expression of hnRNPA1 validated in primary NOTCH1-mutated CLL samples could promote proliferation and inhibit apoptosis in CLL. The expression of hnRNPA1 increased when NOTCH1 signaling was activated by transfection with NOTCH1 intracellular domain (NICD)-overexpressed adenovirus vector and declined after NOTCH1 signaling was inhibited by NOTCH1-shRNA. Higher expression of c-Myc was observed in NICD-overexpressed cells and hnRNPA1 expression was downregulated after applying c-Myc inhibitor 10058-F4. Moreover, in NICD-overexpressed cells, hnRNPA1 expression decreased through c-Myc inhibition.Overexpression of c-Myc-dependent hnRNPA1 could promote proliferation and inhibit apoptosis in NOTCH1-mutated CLL cells, which might partly account for the poor prognosis of patients with NOTCH1 mutation.

Membrane-less organelles and RNP granules are enriched in RNA and RNA-binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear-cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C-terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid-liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane-less organelles, aggregation within phase-separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS-mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS-mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.

Peroxisomes play an essential role in cellular homeostasis by regulating lipid metabolism and the conversion of reactive oxygen species (ROS). Several peroxisomal proteins, known as peroxins (PEXs), control peroxisome biogenesis and degradation. Various mutations in the PEX genes are genetic causes for the development of inheritable peroxisomal-biogenesis disorders, such as Zellweger syndrome. Among the peroxins, PEX1 defects are the most common mutations in Zellweger syndrome. PEX1 is an AAA-ATPase that regulates the recycling of PEX5, which is essential for importing peroxisome matrix proteins. However, the post-transcriptional regulation of PEX1 is largely unknown. Here, we showed that heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) controls PEX1 expression. In addition, we found that depletion of HNRNPA1 induces autophagic degradation of peroxisome, which is blocked in ATG5-knockout cells. In addition, depletion of HNRNPA1 increased peroxisomal ROS levels. Inhibition of the generation of peroxisomal ROS by treatment with NAC significantly suppressed pexophagy in HNRNPA1-deficient cells. Taken together, our results suggest that depletion of HNRNPA1 increases peroxisomal ROS and pexophagy by downregulating PEX1 expression.

The prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation., Graphical Abstract Graphical AbstracthnRNPA1 phase separation is highly salt sensitive. Phase separation of the low-complexity domain (LCD) of hnRNPA1 increases with NaCl. In contrast, phase separation of full-length hnRNPA1 is salt-sensitive. At low NaCl concentrations, electrostatic RRM–LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

Liver fibrosis is a common consequence of chronic liver diseases involved with the activation of hepatic stellate cells (HSCs) and endoplasmic reticulum (ER) stress. Irisin is a small polypeptide hormone that shows beneficial effects on metabolic disorders. The current study aimed to investigate the biological function of irisin on hepatic fibrosis. A mouse model of carbon tetrachloride (CCl4)-induced hepatic fibrosis was established. CCl4-treated mice showed elevated serum levels of AST and ALT, increased collagen accumulation, induced ER stress, and upregulated expressions of pro-fibrotic proteins in the liver compared to the controls. The administration of irisin, however, ameliorated CCl4-induced hepatic fibrosis in both cultured HSCs and mice. PKR-like ER kinase (PERK) is a key component of the ER stress-associated signaling pathway. We found that irisin treatment improved the stability of heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) via regulating the phosphorylation of PERK in mouse livers and isolated HSCs. Also, the knockdown of HNRNPA1 eliminated the hepatoprotective effects of irisin on hepatic fibrosis and ER stress. In summary, this study showed that irisin alleviated ER stress and hepatic fibrosis by inhibiting PERK-mediated HNRNPA1 destabilization, suggesting that irisin may represent a promising therapeutic strategy for patients with liver fibrosis.

Background: A metabolic “switch” from oxidative phosphorylation to glycolysis provides tumor cells with energy and biosynthetic substrates, thereby promoting tumorigenesis and malignant progression. However, the mechanisms controlling this metabolic switch in tumors is not entirely clear. Methods: Clinical specimens were used to determine the effect of SAM68 on lung adenocarcinoma (LUAD) tumorigenesis and metastasis, and mouse models and molecular biology assays were performed to elucidate the function and underlying mechanisms in vitro and in vivo. Results: SAM68 mRNA levels were higher in LUAD tissue than in normal lung tissue, indicating that SAM68 expression is upregulated in LUAD. Patients with LUAD with SAM68high (n = 257) had a higher frequency of tumor recurrence (p = 0.025) and recurrence-free survival (p = 0.013) than did those with SAM68low (n = 257). Patients with SAM68high mRNA levels (n = 257) were at a higher risk for cancer-related death (p = 0.006), and had shorter overall survival (p = 0.044) than did those with SAM68low. SAM68 promotes tumorigenesis and metastasis of LUAD cells in vitro and in vivo by regulating the cancer metabolic switch. SAM68 drives cancer metabolism by mediating alternative splicing of pyruvate kinase (PKM) pre-mRNAs, and promoting the formation of PKM2. Mechanistically, SAM68 increased the binding of the splicing repressor hnRNP A1 to exon 9 of PKM, thereby enhancing PKM2 isoform formation and PKM2-dependent aerobic glycolysis and tumorigenesis. Conclusions: SAM68 promotes LUAD cell tumorigenesis and cancer metabolic programming via binding of the 351-443 aa region of SAM68 to the RGG motif of hnRNP A1, driving hnRNP A1-dependent PKM splicing, contributing to increased oncogene PKM2 isoform formation and inhibition of PKM1 isoform formation. SAM68 is therefore a promising therapeutic target for the treatment of LUAD.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-β2 (Kapβ2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapβ2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapβ2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis., Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) shuttles between the nucleus and cytoplasm to regulate gene expression and RNA metabolism and its low complexity (LC) C-terminal domain facilitates liquid–liquid phase separation and amyloid aggregation. Here, the authors present the cryo-EM structure of amyloid fibrils formed by the hnRNPA1 LC domain, which reveals that the hnRNPA1 nuclear localization sequence forms the fibril core, and they discuss how ALS-causing mutations affect fibril stability.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5′UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

Alternative splicing of pyruvate kinase gene (PKM) results in a higher PKM2/PKM1 ratio that contributes to the Warburg effect and reversing the Warburg effect has opened novel avenues for cancer treatment. miR-206 functions as a tumor suppressor in several types of cancer. However, the effect and underlying mechanisms of miR-206 on the Warburg effect are not yet elucidated. Here, we showed that miR-206 expression was obviously decreased in CRC tissues based on LinkedOmics. A significant decrease in miR-206 expression was negatively correlated with advanced tumor stage, while inversely correlated with overall survival in CRC patients. Ectopic overexpression of miR-206 has dramatically restricted the cell proliferation, glucose consumption and lactate production in CRC cells, whereas transfection of miR-206 inhibitor exhibited the opposite results. Furthermore, miR-206 overexpression induced switching from PKM2 to PKM1 via modulating alternative splicing of PKM gene. The alternative splicing factor hnRNPA1 is identified as the direct functional target of miR-206. Mechanistically, miR-206 overexpression directly targeted hnRNPA1 to suppress PKM2 expression to attenuate Warburg effect and cell proliferation of CRC. Importantly, the restoration of hnRNPA1 expression mostly abrogated the miR-206-meditated Warburg effect. Collectively, these results revealed that the novel miR-206/hnRNPA1/PKM2 axis plays a pivotal role in the Warburg effect to modulate CRC progression.

The full-length mRNAs of the human immunodeficiency virus type-1 (HIV-1), the human T-cell lymphotropic virus type-1 (HTLV-1), and the mouse mammary tumor virus (MMTV) harbor IRESs. The activity of the retroviral-IRESs requires IRES-transacting factors (ITAFs), being hnRNP A1, a known ITAF for the HIV-1 IRES. In this study, we show that hnRNP A1 is also an ITAF for the HTLV-1 and MMTV IRESs. The MMTV IRES proved to be more responsive to hnRNP A1 than either the HTLV-1 or the HIV-1 IRESs. The impact of post-translational modifications of hnRNP A1 on HIV-1, HTLV-1 and MMTV IRES activity was also assessed. Results show that the HIV-1 and HTLV-1 IRESs were equally responsive to hnRNP A1 and its phosphorylation mutants S4A/S6A, S4D/S6D and S199A/D. However, the S4D/S6D mutant stimulated the activity from the MMTV-IRES to levels significantly higher than the wild type hnRNP A1. PRMT5-induced symmetrical di-methylation of arginine residues of hnRNP A1 enabled the ITAF to stimulate the HIV-1 and HTLV-1 IRESs while reducing the stimulatory ability of the ITAF over the MMTV IRES. We conclude that retroviral IRES activity is not only dependent on the recruited ITAFs but also relies on how these proteins are modified at the post-translational level.

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1‑84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross‑reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross‑reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1‑84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross‑reactivity of H1‑84mAb with the glycine (Gly)‑rich domains of hnRNPA1 (195aa‑320aa) and hnRNPA2/B1 (202aa‑349aa) was determined using ELISA blocking experiments. It was concluded that the Gly‑rich domains of these two proteins are heterophilic antigens that cross‑react with influenza virus HA. The association between the heterophilic antigen Gly‑rich domains and the safety of influenza A vaccines remains to be investigated.

Objective Neurodegeneration is thought to be the primary cause of neurological disability in multiple sclerosis (MS). Dysfunctional RNA‐binding proteins (RBPs) including their mislocalization from nucleus to cytoplasm, stress granule formation, and altered RNA metabolism have been found to underlie neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia. Yet, little is known about the role of dysfunctional RBPs in the pathogenesis of neurodegeneration in MS. As a follow‐up to our seminal finding of altered RBP function in a single case of MS, we posited that there would be evidence of RBP dysfunction in cortical neurons in MS. Methods Cortical neurons from 12 MS and six control cases were analyzed by immunohistochemistry for heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and TAR‐DNA‐binding protein‐43 (TDP‐43). Seven distinct neuronal phenotypes were identified based on the nucleocytoplasmic staining of these RBPs. Statistical analyses were performed by analyzing each phenotype in relation to MS versus controls. Results Analyses revealed a continuum of hnRNP A1 and TDP‐43 nucleocytoplasmic staining was found in cortical neurons, from neurons with entirely nuclear staining with little cytoplasmic staining in contrast to those with complete nuclear depletion of RBPs concurrent with robust cytoplasmic staining. The neuronal phenotypes that showed the most nucleocytoplasmic mislocalization of hnRNP A1 and TDP‐43 statistically distinguished MS from control cases (P

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a ubiquitous RNA splicing factor that is overexpressed and prognostically relevant in various human cancer types. To study the impact of hnRNPA1 expression in prostate cancer, we analyzed a tissue microarray containing 17,747 clinical prostate cancer specimens by immunohistochemistry. hnRNPA1 was expressed in normal prostate glandular cells but often overexpressed in cancer cells. hnRNPA1 immunostaining was interpretable in 14,258 cancers and considered strong in 33.4%, moderate in 45.9%, weak in 15.3%, and negative in 5.4%. Moderate to strong hnRNPA1 immunostaining was strongly linked to adverse tumor features including high classical and quantitative Gleason score, lymph node metastasis, advanced tumor stage, positive surgical margin, and early biochemical recurrence (p

Recent studies have demonstrated that various long non‐coding RNAs (lncRNAs) participate in the gastric cancer (GC) development and metastasis. Some lncRNAs exert their regulatory function by interacting with microRNAs. Here we identified a novel lncRNA RP11‐81H3.2 that was highly expressed in the GC tissue and cell lines. RP11‐81H3.2 knockdown significantly inhibited the proliferation, migration, and invasion of GC cells. Mechanistically, we demonstrated that RP11‐81H3.2 directly interacted with miR‐339 while miR‐339 regulated the HNRNPA1 expression by targeting HRRNPA1 3’‐UTR. RP11‐81H3.2‐miR‐339‐HNRNPA1 interaction network regulated the GC cell proliferation, migration, and invasion. Moreover, our results confirmed that RP11‐81H3.2 knockdown suppressed the tumor growth of GC in a xenograft model in vivo. In summary, the results suggest that RP11‐81H3.2 functions as an oncogene in GC and could be utilized as a promising diagnosis and therapeutic marker for GC treatment., LncRNA RP11‐81H3.2 is highly expressed in gastric cancer tissues and cell lines. Knockdown of lncRNA RP11‐81H3.2 suppresses gastric tumor growth in a xenograft tumor model. RP11‐81H3.2/miR‐339/HNRNPA1 interaction network regulates gastric cancer development and progession.