Heterogeneous Ribonucleoprotein Research Articles

#1777 Proteomic profiling of laser capture microdissection kidneys from diabetic nephropathy patients

Abstract Background and Aims Diabetes nephropathy (DN) remains the primary cause of end-stage renal disease (ESRD), warranting equal attention and separate analysis of glomerular, tubular, and interstitial lesions in its diagnosis and intervention. This study aims to identify the specific proteomics characteristics of DN, and assess changes in the biological processes associated with DN. Method 5 patients with DN and 5 healthy kidney transplant donor control individuals were selected for analysis. The proteomic characteristics of glomeruli, renal tubules, and renal interstitial tissue obtained through laser capture microscopy (LCM) were studied using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Results The expression of multiple heat shock proteins, tubulin, and heterogeneous ribonucleoprotein particle in glomeruli and tubules was significantly reduced. Differentially expressed proteins (DEPs) in the glomerulus showed significant enrichment in pathways related to cell junctions and cell movement, including the regulation of actin cytoskeleton and tight junction. DEPs in renal tubules were significantly enriched in glucose metabolism-related pathways, such as glucose metabolism, glycolysis/gluconeogenesis, and the citric acid cycle. Moreover, the glycolysis/gluconeogenesis pathway was a co-enrichment pathway in both DN glomeruli and tubules. Notably, ACTB emerged as the most crucial protein in the PPI analysis of DEPs in both glomeruli and renal tubules. Conclusion By using LCM and the proteomics analysis, we provide insights into the distinctive characteristics of each sub-region of renal tissue, shedding light on the impact of glycolysis metabolic disorder in DN and the potential role of the glomerular cytoskeleton and cell junction.

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway.

Heterogeneous ribonucleoprotein A1 (hnRNPA1) has been reported to enhance the Warburg effect and promote colon cancer (CC) cell proliferation, but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated. To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway. Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b. The relationship between the expression values and the clinicopathological features of the patients was investigated. Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction, while differences in protein expression were analyzed using western blot. Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, and cell cycle and apoptosis were detected using flow cytometric assays. The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay. The Warburg effect was evaluated by glucose uptake and lactic acid production assays. The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls (P < 0.05). Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC, including stage I, II-III, and IV. Furthermore, the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification. HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway, thereby promoting proliferation of HCT116 and SW620 cells. However, the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b, effectively blocking the Warburg effect. These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

Biological functions and clinic significance of SAF‑A (Review).

As one member of the heterogeneous ribonucleoprotein (hnRNP) family, scaffold attachment factor A (SAF-A) or hnRNP U, is an abundant nuclear protein. With RNA and DNA binding activities, SAF-A has multiple functions. The present review focused on the biological structure and different roles of SAF-A and SAF-A-related diseases. It was found that SAF-A maintains the higher-order chromatin organization via RNA and DNA, and regulates transcription at the initiation and elongation stages. In addition to regulating pre-mRNA splicing, mRNA transportation and stabilization, SAF-A participates in double-strand breaks and mitosis repair. Therefore, the aberrant expression and mutation of SAF-A results in tumors and impaired neurodevelopment. Moreover, SAF-A may play a role in the anti-virus system. In conclusion, due to its essential biological functions, SAF-A may be a valuable clinical prediction factor or therapeutic target. Since the role of SAF-A in tumors and viral infections may be controversial, more animal experiments and clinical assays are needed.

SARS-CoV-2 infection engenders heterogeneous ribonucleoprotein interactions to impede translation elongation in the lungs.

Translational regulation in tissue environments during in vivo viral pathogenesis has rarely been studied due to the lack of translatomes from virus-infected tissues, although a series of translatome studies using in vitro cultured cells with viral infection have been reported. In this study, we exploited tissue-optimized ribosome profiling (Ribo-seq) and severe-COVID-19 model mice to establish the first temporal translation profiles of virus and host genes in the lungs during SARS-CoV-2 pathogenesis. Our datasets revealed not only previously unknown targets of translation regulation in infected tissues but also hitherto unreported molecular signatures that contribute to tissue pathology after SARS-CoV-2 infection. Specifically, we observed gradual increases in pseudoribosomal ribonucleoprotein (RNP) interactions that partially overlapped the trails of ribosomes, being likely involved in impeding translation elongation. Contemporaneously developed ribosome heterogeneity with predominantly dysregulated 5 S rRNP association supported the malfunction of elongating ribosomes. Analyses of canonical Ribo-seq reads (ribosome footprints) highlighted two obstructive characteristics to host gene expression: ribosome stalling on codons within transmembrane domain-coding regions and compromised translation of immunity- and metabolism-related genes with upregulated transcription. Our findings collectively demonstrate that the abrogation of translation integrity may be one of the most critical factors contributing to pathogenesis after SARS-CoV-2 infection of tissues.

Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

New Insights into Dyskerin-CypA Interaction: Implications for X-Linked Dyskeratosis Congenita and Beyond.

The highly conserved family of cyclophilins comprises multifunctional chaperones that interact with proteins and RNAs, facilitating the dynamic assembly of multimolecular complexes involved in various cellular processes. Cyclophilin A (CypA), the predominant member of this family, exhibits peptidyl-prolyl cis-trans isomerase activity. This enzymatic function aids with the folding and activation of protein structures and often serves as a molecular regulatory switch for large multimolecular complexes, ensuring appropriate inter- and intra-molecular interactions. Here, we investigated the involvement of CypA in the nucleus, where it plays a crucial role in supporting the assembly and trafficking of heterogeneous ribonucleoproteins (RNPs). We reveal that CypA is enriched in the nucleolus, where it colocalizes with the pseudouridine synthase dyskerin, the catalytic component of the multifunctional H/ACA RNPs involved in the modification of cellular RNAs and telomere stability. We show that dyskerin, whose mutations cause the X-linked dyskeratosis (X-DC) and the Hoyeraal-Hreidarsson congenital ribosomopathies, can directly interact with CypA. These findings, together with the remark that substitution of four dyskerin prolines are known to cause X-DC pathogenic mutations, lead us to indicate this protein as a CypA client. The data presented here suggest that this chaperone can modulate dyskerin activity influencing all its partecipated RNPs.

Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNA.

A human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments.

Knockdown of hnRNPAB reduces the stem cell properties and enhances the chemosensitivity of human colorectal cancer stem cells.

Heterogeneous ribonucleoproteinAB (hnRNPAB) is one of the main members of the nuclear heterogeneous ribonucleoprotein family and plays a crucial role in the occurrence and development of tumours. A previous study by the authors demonstrated that hnRNPAB was highly expressed in colorectal cancer tissues and was closely associated with a poor prognosis of patients. However, the contribution of hnRNPAB to the tumorigenesis and drug resistance of colorectal cancer (CRC) stem cells (CSCs) remains elusive. The aim of the present study was thus to examine whether hnRNPAB can enhance the characteristics of colorectal CSCs and chemotherapeutic drug resistance by altering the cell cycle and the apoptosis of colorectal CSCs. The results revealed that the expression of hnRNPAB in colorectal CSCs was increased compared with that in their parental cells. The knockdown of hnRNPAB reduced the sphere formation of and the levels of CSC markers in colorectal CSCs, enhanced sensitivity to 5‑fluorouracil and oxaliplatin chemotherapy and increased apoptosis. Taken together, these data indicate the role of hnRNPAB in maintaining CSC properties and provide a novel therapeutic target for the treatment of CRC and particularly, drug resistance.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

Bta-miR-206 and a Novel lncRNA-lncA2B1 Promote Myogenesis of Skeletal Muscle Satellite Cells via Common Binding Protein HNRNPA2B1.

Skeletal muscle satellite cells (MuSCs) can proliferate, differentiate, and self-renew, and can also participate in muscle formation and muscle injury repair. Long noncoding RNAs (lncRNAs) can play an important role with the RNA binding protein and microRNAs (miRNAs) to regulate the myogenesis of bovine MuSCs, however, its molecular mechanism is still being explored. In this study, differentially expressed 301 lncRNAs were identified during the myogenic differentiation of cells based on an in vitro model of induced differentiation of bovine MuSCs using RNA sequencing (RNA-seq). Based on the ability of miR-206 to regulate myogenic cell differentiation, a new kind of lncRNA-lncA2B1 without protein-coding ability was found, which is expressed in the nucleus and cytoplasm. Subsequently, lncA2B1 inhibited cell proliferation by downregulating the expression of the proliferation marker Pax7 and promoted myogenic differentiation by upregulating the expression of the differentiation marker MyHC, whose regulatory function is closely related to miR-206. By RNA pulldown/LC-MS experiments, heterogeneous ribonucleoprotein A2/B1 (HNRNPA2B1), and DExH-Box Helicase 9 (DHX9) were identified as common binding proteins of lncA2B1 and miR-206. Overexpression of lncA2B1 and miR-206 significantly upregulated the expression level of HNRNPA2B1. Downregulation of HNRNPA2B1 expression significantly decreased the expression level of the differentiation marker MyHC, which indicates that miR-206 and lncA2B1 regulate myogenic differentiation of bovine MuSCs by acting on HNRNPA2B1. This study screened and identified a novel lncRNA-lncA2B1, which functions with miR-206 to regulate myogenesis via the common binding proteins HNRNPA2B1. The results of this study provide a new way to explore the molecular mechanisms by which lncRNAs and miRNAs regulate muscle growth and development.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

LncRNA Ctcflos modulates glucocorticoid receptor-mediated induction of hepatic phosphoenolpyruvate carboxykinase in mice

AimsPhosphoenolpyruvate carboxykinase (Pck1) is a key enzyme that catalyzes an irreversible step of hepatic gluconeogenesis; however, the mechanism underlying Pck1 regulation is not fully understood. In the present study, we investigated the role of Ctcflos, a long non-coding RNA (lncRNA) located in the mouse Pck1 enhancer region, in the transcriptional regulation of the Pck1 gene. Main methodsRapid amplification of cDNA ends (RACE) was done to identify the precise structures of Ctcflos isotypes. We analyzed the DNA binding signals of GR, hnRNPL, and histone marks by ChIP-qPCR assays. We used the antisense oligonucleotide (ASO) for suppression of Ctcflos and adenovirus encoding Ctcflos was injected into mice via the tail vein to overexpress Ctcflos in the liver. We performed RNA pull-down assays to search for Ctcflos-binding proteins. Key findingsWe identified the precise structures of five different hepatic Ctcflos isotypes. The induction of Ctcflos was dependent on the glucocorticoid receptor (GR) in the livers of fasted or dexamethasone-administered mice. Overexpression of Ctcflos increased the expression of Pck1, whereas knockdown of Ctcflos suppressed the transcript and protein expression of Pck1. Ctcflos bound the heterogeneous ribonucleoprotein L (hnRNPL), which interacts with GR. Knockdown of hnRNPL upregulated the Pck1 gene. The interaction between Ctcflos and hnRNPL interfered the binding of GR to hnRNPL, which subsequently increased GR recruitment in the Pck1 enhancer region. SignificanceTargeting Ctcflos may provide a new insight into the development of alternative treatments for hyperglycemia by offering specific regulation of hepatic Pck1 gene expression.

Affinity-Based Interactome Analysis of Endogenous LINE-1 Macromolecules.

During their proliferation and the host's concomitant attempts to suppress it, LINE-1 (L1) retrotransposons give rise to a collection of heterogeneous ribonucleoproteins (RNPs); their protein and RNA compositions remain poorly defined. The constituents of L1-associated macromolecules can differ depending on numerous factors, including, for example, position within the L1 life cycle, whether the macromolecule is productive or under suppression, and the cell type within which the proliferation is occurring. This chapter describes techniques that aid the capture and characterization of protein and RNA components of L1 macromolecules from tissues that natively express them. The protocols described have been applied to embryonal carcinoma cell lines that are popular model systems for L1 molecular biology (e.g., N2102Ep, NTERA-2, and PA-1 cells), as well as colorectal cancer tissues. N2102Ep cells are given as the use case for this chapter; the protocols should be applicable to essentially any tissue exhibiting endogenous L1 expression with minor modifications.

Assay Development and Small Molecule Screens for Inhibitors of an RNA Binding Protein Implicated in Hematologic Malignancies

RNA-binding proteins (RBPs) play a critical role in regulating RNA splicing, polyadenylation, and translation. RBPs typically contain discrete RNA-binding domains (RBDs) that bind to specific nucleotide motifs. Given their important role in regulating gene expression, RBPs are often mutated or aberrantly expressed in a variety of malignancies, particularly hematological cancers. As a result, RBPs appear to be excellent therapeutic targets. However, given that most RBPs lack enzymatic activity, it has been challenging to develop targeted therapies. Over the past several years we and others have used genetic and molecular biology approaches to understand the consequence of aberrant expression of the poly-C RBP, heterogeneous ribonucleoprotein K (hnRNP K), and determine the mechanisms by which it drives hematologic malignancies (Cancer Cell, 2015; NEJM, 2013, JNCI, 2020; and BioRxiv, 2021). To develop a pharmacologic strategy to target hnRNP K's RNA binding activities, we developed a robust fluorescent anisotropy (FA) assay to evaluate hnRNP K's ability to bind target transcripts that are critical for tumorigenesis; such as, BRD4, MYC, RUNX1, and 7SK. We next performed small molecule screens using ~80,000 compounds and identified 34 compounds that inhibited hnRNP K's ability to bind RNA targets. To gain a deeper understanding of whether these compounds directly interact with hnRNP K to block its RNA binding functions, we performed SYPRO-orange thermal shift assays. Here we identified compounds that bound hnRNP K directly and altered hnRNP K thermostability. To evaluate association rate, dissociation rate, and KD constants, we next performed SPR assays. These experiments identified compounds bound hnRNP K with affinities between (350nM and 20uM). We next performed cell-based studies, such as CETSA. Here we identified compounds that crossed the plasma membrane, directly engaged hnRNP K in vivo, and significantly stabilized hnRNP K. Molecular analyses revealed that cells treated with these lead compounds have reduced c-Myc expression, a direct mRNA target of hnRNP K. To develop a granular understanding of how these hnRNP K-small molecule interactions impact growth and cell survival, we performed high-throughput cell-based assays. These studies demonstrated that cells with inducible hnRNP K overexpression were sensitive to these small molecules. To begin medicinal chemistry approaches and to understand each compound's chemical requirements for binding to hnRNP K, we scanned molecules for reactive moieties. Here, we identified two compounds possessing amide groups within two carbons of either a furan or pyrrole moiety. These compounds, #20 and #23, respectively, were then chemically modified and FA assays were repeated in the presence of target mRNAs. Here, we observed that both the furan and isoxazole ring of compound #20 and pyrrole rings of compound #23 are critical for hnRNP K interactions, while the cyclopropane moiety on compound #23 is dispensable. Further analyses revealed additional structural analogs with either increased or decreased affinities and these results have been critical to our understanding of the importance of each moiety. Currently, we are using cryo-EM to evaluate positional interactions between hnRNP K (hnRNP K's RBDs) and these small molecules. These results will be used to further our medicinal chemistry approaches to generate more potent analogs. Collectively, our results demonstrate that hnRNP K functions can be directly inhibited by small molecules that block its RNA binding functions and demonstrates that the development of effective therapies for RBPs is possible. 1. hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies. Cancer Cell, 2015. 28(4): p. 486-499. 2. Cancer Genome Atlas Research Network, Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013 368(22):2059-74. 3. Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas. JNCI: Journal of the National Cancer Institute, 2020. 4. Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing. BioRxiv. doi: https://doi.org/10.11.02.05.429385, 2021 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heterogeneous ribonucleoprotein D selectively regulates the expression of genes in the immune system

AbstractThe immune response is critical for the elimination of pathogens. However, uncontrolled immune responses can lead to tissue damage and inflammatory diseases. The generation of an immune response requires the activation, differentiation and proliferation of various immune cells and the production of cytokines that are regulated directly or indirectly by multiple pathways, including posttranscriptional regulation by RNA‐binding proteins (RBPs). Heterogeneous ribonucleoprotein D (hnRNPD), an RBP that is also known as AU‐rich elements/poly(U)‐binding/degradation factor 1 (AUF1), can regulate the expression of various target RNAs, including mRNAs, miRNAs, lncRNAs, by binding to their Au‐rich elements (AREs) or U‐/GU‐rich sequences. The target RNAs of hnRNPD range from cytokines, inflammatory receptors and growth factors to G protein‐coupled receptors; thus, hnRNPD influences various physiological processes, including the immune response. In addition, hnRNPD is strongly expressed in multiple lymphocytic tissues, suggesting that it may play an important role in the immune system. In this review, we discuss the roles of hnRNPD in the immune system and autoimmune diseases.

Tumor-Promoting Actions of HNRNP A1 in HCC Are Associated with Cell Cycle, Mitochondrial Dynamics, and Necroptosis.

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.

Characterization and functional analysis of chicken dsRNA binding protein hnRNPU

In mammals, heterogeneous ribonucleoprotein U (hnRNPU), also named as nuclear matrix protein-nuclear scaffold attachment factor (SAFA), was originally identified as a DNA/RNA interactor protein. It has been reported that human hnRNPU facilitates IFN-β generation after vesicular stomatitis virus (VSV) infection. Nevertheless, the role of chicken hnRNPU (chhnRNPU) in IFN-β regulation as well as in infectious bursal diseases virus (IBDV) replication is still unclear. Here, we found that chhnRNPU inhibits IFN-β production via interacting with MDA5 and MAVS, and facilitates IBDV replication via associating with genomic dsRNA of IBDV. Firstly, chicken hnRNPU (chhnRNPU) was widely expressed in different tissues of chickens and was distributed in the nucleus of DF-1 cells. Overexpression of chhnRNPU significantly suppresses IFN-β promoter activities induced by MDA5 and MAVS. Additionally, immunoprecipitated by dsRNA antibodies, which followed LC-MS analysis demonstrate that chhnRNPU is a partner of viral genomic dsRNA. chhnRNPU is translocated from nucleus to cytosol to co-localize with replication complex of IBDV after IBDV infection. Over-expression of chhnRNPU significantly promotes IBDV replication, which was determined by western blotting, qRT-PCR and TCID50 assay. Furthermore, knock down chhnRNPU by siRNA remarkably facilitates IFN-β production, and inhibits IBDV proliferation. These data collectively reveal that chhnRNPU positively regulates IBDV replication via negatively regulating IFN-β response.

PKCq modulates LKB1 isoforms downstream of IL-6 signaling to mediate iTreg-Th17 cell plasticity

Abstract Following activation, CD4 T cells undergo metabolic and transcriptional changes responding to external cues and differentiate into T helper (Th) cells. This differentiation process was perceived to be terminal. However, it has been shown that T cells exhibit plasticity between subsets in highly inflammatory environments. This phenomenon is prevalent in autoimmune conditions, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCq) is a serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg polarization. The tumor suppressor liver kinase B1 (LKB1) encoded by Stk11, a serine/threonine kinase, enforces energy-preserving mechanisms such as fatty acid oxidation, a major energy source for Tregs. Recent studies demonstrate loss of LKB1 disrupts Treg survival and function. Stk11 can be alternatively spliced into a short variant by the inclusion of a cryptic exon. However, specificity of LKB1 isoforms influencing Th plasticity has not been elucidated. Our study shows that the expression of the Stk11 short splice variant (Stk11S) is upregulated in Th17 compared to induced (i)Tregs. We demonstrate that, in Th17 cells heterogeneous ribonucleoprotein L-like (hnRNPLL), a splicing factor promoting cryptic exon inclusion, binds to transcripts of Stk11 and correlates with increased expression of Stk11S. We further show in iTregs exposed to IL-6, Prkcq, Stk11S, and Rorc are upregulated, suggesting iTreg-Th17 plasticity is induced in response to IL-6 resulting in Stk11 splicing downstream of PKCq. Thus, PKCq may destabilize iTreg programming through its regulation of Stk11 splicing under pro-inflammatory conditions. Supported by grants from the department of defense.

NF\u03baB (RelA) mediates transactivation of hnRNPD in oral cancer cells

Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5ʹ flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (−1358/−1347) motif and 99% reduction after the deletion of second motif (−1052/−1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen’s Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.