Heterogeneous Ribonucleoprotein Research Articles

Cyclic AMP-Dependent Protein Kinase Phosphorylates TDP-43 and Modulates Its Function in Tau mRNA Processing.

Trans-active response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved and ubiquitously expressed nuclear protein. As a member of heterogeneous ribonucleoproteins, TDP-43 plays pivotal roles in mRNA processing. We recently found that TDP-43 promoted tau mRNA instability via acting on the 3'-untranslated region of its mRNA and enhanced tau exon 10 inclusion. TDP-43 is a phospho-protein. The function and the pathological aggregation of TDP-43 are regulated by the phosphorylation. In the present study, we determined phosphorylation of TDP-43 by cyclic AMP-dependent protein kinase (PKA). We found that TDP-43 was co-immunoprecipitated by and co-localized with PKA in the nucleus. PKA phosphorylated TDP-43 at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells. Phosphorylation of TDP-43 at these sites enhanced mutually their phosphorylation by PKA in vitro and in cultured cells. Overexpression of PKA suppressed TDP-43's activity in promoting tau mRNA instability and tau exon 10 inclusion. These findings shed light on the role of PKA in phosphorylation and function of TDP-43. Downregulation of PKA signaling in AD brain may attenuate the impact of TDP-43 pathology in tau pathogenesis.

Effect of sevoflurane combined with propofol anesthesia on postoperative expression of hnRNPA2 in brain tissues of rats with mild cognitive impairment

Objective To evaluate the effect of sevoflurane combined with propofol anesthesia on the postoperative expression of nuclear heterogeneous ribonucleoprotein A2 (hnRNPA2) in brain tissues of rats with mild cognitive impairment (MCI). Methods Male Sprague-Dawley rats, aged 16-18 months, were anesthetized with pentobarbital sodium.MCI was induced by ligation of bilateral common carotid arteries after anesthesia.Forty-eight rats with MCI were divided into 4 groups (n=12 each) using a random number table method: sham operation group (SH group), sevoflurane anesthesia group (S group), propofol anesthesia group (P group), and sevoflurane combined with propofol anesthesia group (SP group). Group S inhaled 3% sevoflurane.Propofol was intravenously infused at a rate of 40 mg·kg-1·h-1 in group P. In group SP, 1.7% sevoflurane was inhaled, and propofol 20 mg·kg-1·h-1 was intravenously infused.The anesthesia time was 3 h in the three groups.After disappearance of eyelash reflex, open reduction and internal fixation was performed after tibial fracture was induced.Y-maze test was performed at 7 days after operation, and the percentage of time of staying at novel arm was calculated.The open field test was performed, and the total activity distance and time of staying at the central region were recorded.Then the rats were sacrificed, and brain tissues were obtained for determination of the expression of hnRNAP2 and γ-aminobutyric acid receptor A1 subunit (GABAA-α1) in hippocampus by immunofluorescence and Western blot, respectively. Results Compared with SH group, the percentage of time of staying at novel arm was significantly decreased, the expression of hnRNPA2 in the hippocampus was up-regulated, and the expression of GABAA-α1 was down-regulated in S and P groups, and the expression of hnRNPA2 in the hippocampus was up-regulated (P 0.05). Compared with S group or P group, the percentage of time of staying at novel arm was significantly increased, the expression of hnRNPA2 in the hippocampus was down-regulated, and the expression of GABAA-α1 was up-regulated in SP group (P 0.05). Conclusion The mechanism by which sevoflurane combined with propofol anesthesia does not aggravate the postoperative cognitive dysfunction may be related to up-regulating the expression of hnRNPA2 in brain tissues and maintaining GABAA-α1 stable in rats with MCI. Key words: Anesthetics, inhalation; Propofol; Cognition disorders; Heterogeneous nuclear ribonucleoprotein A2

Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.

Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.

Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U.

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

Transcriptome protection by the expanded family of hnRNPs

ABSTRACTThe family of heterogeneous ribonucleoproteins (hnRNPs) have multiple functions in RNA metabolism. In recent years, several hnRNPs have also been shown to be essential for the maintenance of transcriptome integrity, by preventing intronic cryptic splicing signals from mis-splicing of many endogeneous pre-mRNA transcripts. Here we discuss the possibility for a general role of this family of proteins and their expansion in transcriptome protection.

Ubiquilin 2 modulates ALS/FTD-linked FUS–RNA complex dynamics and stress granule formation

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.

Erratum: Prognostic value of heterogeneous ribonucleoprotein A1 expression and inflammatory indicators for patients with surgically resected hepatocellular carcinoma: Perspectives from a high occurrence area of hepatocellular carcinoma in China.

[This corrects the article DOI: 10.3892/ol.2018.9079.].

Heterogeneous ribonucleoprotein F regulates YAP expression via a G-tract in 3′UTR

The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3′UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3′UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.

PTB/nPTB: master regulators of neuronal fate in mammals

PTB was initially discovered as a polypyrimidine tract-binding protein (hence the name), which corresponds to a specific RNA-binding protein associated with heterogeneous ribonucleoprotein particle (hnRNP I). The PTB family consists of three members in mammalian genomes, with PTBP1 (PTB) expressed in most cell types, PTBP2 (also known as nPTB or brPTB) exclusively found in the nervous system, and PTBP3 (also known as ROD1) predominately detected in immune cells. During neural development, PTB is down-regulated, which induces nPTB, and the expression of both PTB and nPTB becomes diminished when neurons mature. This programed switch, which largely takes place at the splicing level, is critical for the development of the nervous system, with PTB playing a central role in neuronal induction and nPTB guarding neuronal maturation. Remarkably, sequential knockdown of PTB and nPTB has been found to be necessary and sufficient to convert non-neuronal cells to the neuronal lineage. These findings, coupled with exquisite understanding of the molecular circuits regulated by these RNA-binding proteins, establish a critical foundation for their future applications in regenerative medicine.

Prognostic value of heterogeneous ribonucleoprotein A1 expression and inflammatory indicators for patients with surgically resected hepatocellular carcinoma: Perspectives from a high occurrence area of hepatocellular carcinoma in China.

Heterogeneous ribonucleoproteinA1 (hnRNPA1) is a documented tumor biomarker known to be aberrantly expressed in a number of types of human cancer. However, to the best of our knowledge, its prognostic value for surgically resected HCC (RHCC) in the high incidence areas of China has not been described; the association between hnRNPA1 expression, pre-operative neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) is also not understood. In the present study, hnRNPA1 expression was retrospectively measured in two independent cohorts of patients with hepatocellular carcinoma (HCC) who underwent surgery to remove the primary cancer at one center in Fujian, an area with a high incidence of HCC in China. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunohistochemistry (IHC) were used to quantify hnRNPA1 expression in RHCC tissues. The survival curves were plotted using the Kaplan-Meier method, and the prognostic significance of hnRNPA1, NLR and PLR was analyzed using the log-rank test. The relevant prognostic factors were identified by multivariate Cox regression analysis. RT-qPCR and western blotting revealed that hnRNPA1 was upregulated in HCC tissues (P<0.001), and particularly overexpressed in tumor tissues of patients with recurrent HCC (P<0.001) (cohort 1; 54 patients). Differential hnRNPA1 expression was measured in 426HCC tissues with IHC; 259 exhibited high hnRNPA1 expression and 167 exhibited low expression. High hnRNPA1 expression was significantly associated with Tumor-Node-Metastasis stage (P=0.024), tumor size (P=0.027), vascular invasion (P<0.001), Edmonson grade (P<0.001), pre-operative serum α-fetoprotein (AFP) (P<0.001), NLR (P<0.001) and PLR (P<0.001). In addition, multivariate Cox regression analysis confirmed that high hnRNPA1 expression was associated with relapse-free survival (RFS; HR, 0.685; 95% CI, 0.506–0.928; P=0.015) and overall survival (OS; HR, 0.629; 95% CI, 0.454–0.871; P=0.005). Multivariate analysis confirmed that higher pre-operative serum AFP had an unfavorable impact on RFS (HR, 1.350; 95% CI, 1.006–1.811; P=0.045) and OS (HR=1.564; 95% CI, 1.151–2.126; P=0.004), while higher pre-operative NLR had an unfavorable impact on OS (HR, 1.758; 95% CI, 1.161–2.661; P=0.008) (cohort 2;426 patients). The expression of hnRNPA1 was also positively correlated with NLR (Spearman's correlation; r=0.122, P=0.012) and PLR (Spearman's correlation; r=0.140, P=0.004). In conclusion, high hnRNPA1 expression was revealed as prognostic for poor survival in patients with RHCC, and detection of hnRNPA1 protein in tumor tissues demonstrated potential in estimating survival for patients with RHCC in areas with high incidence rates. Furthermore, the combination of high hnRNPA1 expression and pre-operative serum AFP levels (>400 µg) proved to be a good diagnostic and prognostic biomarker for this specific population of patients. Finally, a correlation may also exist between hnRNPA1 expression and other markers of systemic inflammation.

Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions

ABSTRACTLong noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Abstract 4434: Contribution of heterogenous ribonucleoprotein A18 in prostate cancer progression

Abstract Prostate cancer is the third leading cause of cancer deaths among men in the United States, with recurrent metastasis being the predominant cause of mortality. In spite of the significant progress made in the past half century with hormonal therapy for metastatic prostate cancer, a majority eventually progresses to a lethal stage of the disease commonly known as castration-resistant prostate cancer. New treatment strategies are therefore needed. Previous work from our lab has identified the RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) as a new regulator of protein translation in cancer cells. While its localization is predominantly nuclear, hnRNP A18 translocates out to the cytosol in response to cellular stress like hypoxia. Immunohistochemistry data from tissue microarrays reveals increased expression of hnRNP A18 in prostate hyperplasia, and even greater overexpression in prostate adenocarcinoma as compared to the surrounding normal tissue. Furthermore, a greater percentage of hnRNP A18 is seen to shift from the nucleus to the cytosol as the disease progresses. In the cytosol, hnRNP A18 recognizes and binds to a specific 51 nucleotide RNA motif in the 3' untranslated region of targeted transcripts, stabilizing them and enhancing their translation. Transcripts targeted by hnRNP A18 confer tumor growth advantages. The down-regulation of hnRNP A18 was seen to reduce cancer cell proliferation, migration, and invasion. Moreover, down-regulation of hnRNP A18 significantly reduces prostate cancer growth in a mouse tumor xenograft model. Taken together, our data suggests that hnRNP A18 could be a potentially new target to stop or limit prostate cancer progression. Citation Format: Elizabeth Tsuying Chang, Eun Yong Choi, Palak R. Parekh, France Carrier. Contribution of heterogenous ribonucleoprotein A18 in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4434.

Heterogeneous ribonucleoprotein M influences specific cytokine outcomes upon Salmonella Typhimurium infection

Abstract A complex and dynamic regulatory system is needed to fight infection while also preventing induction of inflammatory disease. In macrophages, much is known about how pathogens modulate the innate immune transcriptional response. However, the significance of alternative splicing and manipulation of splicing factors upon infection remains unclear. We have identified several splicing factors that are differentially phosphorylated upon Salmonella Typhimurium bacterial infection, strongly suggesting an important role for RNA processing in the regulation of innate immune responses. A major family of identified phosphorylated splicing factors was the hnRNP (heterogeneous ribonucleoprotein) RNA binding factors, which contribute to multiple aspects of nucleic acid metabolism. Upon LPS stimulation and Salmonella Typhimurium infection, we have found that one of these proteins, hnRNPM, modulates expression of an inflammatory cytokine, IL-6. We have also identified hnRNPM binding sites within the introns of IL-6. We are currently investigating how hnRNPM localizes to chromatin, how hnRNPM intron recognition regulates IL-6 transcript levels, and how the phosphorylation of hnRNPM influences these processes.

The RNA-Binding Protein PCBP1 Functions as a Tumor Suppressor in Prostate Cancer by Inhibiting Mitogen Activated Protein Kinase 1

Background/Aims: Poly r(C) binding protein (PCBP) 1 or heterogeneous ribonucleoprotein (hnRNP) E1 is a RNA binding protein functional in multiple biological processes. In prostate cancer (PCa), PCBP1 loss was shown to be involved with increased stemness in PCacells; however, the underlying mechanism remains unclear. Method: The role of PCBP1 in prostate tumor formationwas determined by xenograft assays. Immunoprecipitationand mass spectrometry were performed to find the pathways altered after PCBP1 knockdown. Cell proliferation, migration, invasion, and soft agar colony formationassays and xenograft assays were used to determine the role of target protein pathogenesis regulation and formation of PCa. QRT-PCR was performedto quantify relative mRNA expression. Results: The expression of mitogen activated protein kinase 1 (MAPK1) or extracellular signal regulated kinase 2 (ERK2) was increased following PCBP1 loss. Attenuation of MAPK1 inhibited in vitro and in vivo tumorigenicity and metastasis in PCa cell line, PC3. Overexpression of MAPK1 in the PC3 cells increased the tumorigenicity and metastasis. Analysis of PCBP1 and MAPK1 mRNA levels in 25 PCa patients compared to tumor-adjacent normal tissue confirmed an inverse correlation between PCBP1 and MAPK1 expression. Conclusions: PCBP1 can act as a suppressor of tumor in prostate epithelial cells by inhibiting MAPK1 expression.

Effect of heterogeneous ribonucleoprotein A2/B1 on lipopolysaccharide-induced permeability of vascular endothelial cells

Objective To investigate the effect of heterogeneous ribonucleoprotein A2/B1 (hnRNP A2/B1) on the human umbilical vein endothelial cells (HUVECs) permeability induced by lipopolysaccharide(LPS). Methods HUVECs were used in this study, small interfering RNA (siRNA) with hnRNP A2/B1 gene silencing was screened. The samples were divided into four groups: NC siRNA group, hnRNP A2/B1 siRNA group, NC siRNA-LPS group and hnRNP A2/B1 siRNA-LPS group. The transendothelial electrical resistance (TEER) of HUVECs was detected at 0, 6, 12 h and 24 h. The expression of vascular endothelial cadherin (VE-cadherin) and intercellular adhesion molecule-1 (ICAM-1) were measured at 12 h. Results After LPS treatment, the TEER values of HUVECs in hnRNP A2/B1 siRNA-LPS group was significantly decreased compared with values of NC siRNA-LPS group at 12 h (P<0.05). The expression level of VE-cadherin in hnRNP A2/B1 siRNA-LPS group was lower than the level in the NC siRNA-LPS group, and the expression level of ICAM-1 was increased in hnRNP A2/B1 siRNA-LPS group compared with the level of NC siRNALPS group(P<0.05). Conclusions hnRNP A2/B1 may play an important role in vascular endothelial permeability via regulating the expression of VE-cadherin and ICAM-1, thus protecting vascular endothelial barrier function. Key words: Heterogeneous ribonucleoprotein A2/B1; Lipopolysaccharide; Endothelial permeability; Vascular endothelial cadherin

Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.

The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.

Osteopontin-c isoform levels are associated with SR and hnRNP differential expression in ovarian cancer cell lines.

Osteopontin-c splicing isoform activates ovarian cancer progression features. Imbalanced expression of splicing factors from serine/arginine -rich and heterogeneous ribonucleoproteins families has been correlated with the generation of oncogenic splicing isoforms. Our goal was to investigate whether there is any association between the transcriptional patterns of these splicing factors in ovarian cells and osteopontin-c expression levels. We also aimed to investigate the occurrence of these splicing factors binding sites inside osteopontin exon 4 and adjacent introns. To test associations between osteopontin-c and splicing factors expression patterns, we used an in vitro model in which OVCAR-3 cells overexpressing osteopontin-c (OVCAR-3/OPNc++) presented higher transcriptional levels of osteopontin-c than two other ovarian carcinoma cells (TOV-112D, SKOV-3) and ovarian non-tumoral cell lines (IOSE 364 and IOSE 385). The transcriptional levels of osteopontin-c, serine/arginine-rich, and hnRNP factors were evaluated using real-time polymerase chain reaction. Human Splice Finder software was used to search for putative splicing factor binding sites in osteopontin genomic regions. OVCAR-3/OPNc++ cells presented higher transcriptional levels of hnRNP than serine/arginine-rich when compared to TOV-112D, SKOV-3, and IOSE cells. TOV-112D and SKOV-3 cells also overexpressed hnRNP in relation to serine/arginine-rich transcripts. Putative binding sites for these splicing factors have been predicted on osteopontin exon 4 and their upstream and downstream intronic regions. Our data showed that higher osteopontin-c expression levels are associated with a predominance of hnRNP in relation to serine/arginine-rich transcripts and that osteopontin exon 4 and adjacent intronic sequences contain predicted binding sites for some of these tested splicing factors. In conclusion, differential expression of these splicing factors in ovarian cancer cells could be one of the putative mechanisms leading to aberrant splicing of the osteopontin primary transcript. Future work, aiming to control ovarian cancer progression by downregulating osteopontin-c levels, could include strategies that also regulate heterogeneous ribonucleoproteins and serine/arginine-rich expression levels in order to modulate osteopontin splicing.

Abstract 4478: Characterization of the biochemical interaction of hnRNP A18 to thioredoxin transcript

Abstract Introduction: The RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) is a protein translation regulator found to be elevated in many cancers. Previous work from our lab has shown that under stress conditions, hnRNP A18 is able to bind and regulate the translation of a group of mRNAs, including thioredoxin (TRX), a redox-regulating protein implicated in promoting tumor progression. Additionally, the formation of functional hnRNP A18:TRX complexes increases de novo protein synthesis of TRX through the stabilization of its mRNA transcript. It has been reported that like most RNA-binding proteins, hnRNP A18 is a modular protein and is uniquely composed of a single N-terminal RNA binding domain (RBD) and a C-terminal arginine-glycine rich (RGG) domain. The RBD is able to dictate specificity and affinity of substrate binding, while methylation of the arginine residues in the RGG domain contributes to its RNA binding capability. The observed translational regulation of TRX by hnRNP A18 merits further studies into understanding the physical interaction between the two molecules. Methods/Results: To determine the region which hnRNP A18 interacts with thioredoxin, stable cell lines overexpressing the RBD, RGG or full length (FL) domains of hnRNP A18 were generated in human melanoma cells. Ribonucleoprotein immunoprecipitation assays were performed using specific antibodies to either the RBD or RGG domain epitope. Reverse transcription PCR data reveals the presence of TRX mRNA in the resultant immunoprecipitated materials, indicating that both the RBD and RGG domain of hnRNP A18 protein is able to independently bind TRX mRNA. Maximum binding was observed when both RBD and RGG regions were present. Furthermore, hnRNP A18-GST recombinant proteins were engineered to determine the exact region which hnRNP A18 binds to on its target mRNAs. TRX mRNA was systematically deleted and increasing amounts of hnRNP A18-GST protein was added to determine binding capability. Northwestern analysis reveals that hnRNP A18 binds to its consensus motif in the TRX 3’UTR and to a new 19 nucleotide motif downstream of the consensus site. Conclusion: Heterogenous ribonucleoprotein A18 is a stress-activated RNA-binding protein that functions in the translational regulation of TRX. Although the RBD and RGG domains are each independently able to physically interact with TRX, both regions are necessary for maximal binding. Furthermore, a new 19 nucleotide motif located downstream of the hnRNP A18 consensus motif was also found to be sufficient for binding. The binding interface between RNA-binding proteins and their target mRNAs hold significant importance in understanding how the two molecules interact and will offer new insights into developing novel drug therapeutics that disrupt ribonucleoprotein formation. Citation Format: Elizabeth Tsuying Chang, Palak Parekh, Eun Yong Choi, Ruiqing Yang, France Carrier. Characterization of the biochemical interaction of hnRNP A18 to thioredoxin transcript [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2017-4478

High expression of hnRNPAB/Kap1 together promote poor prognosis in HCC

Objective: To further understand the interaction protein spectrum of heterogeneous ribonucleoprotein AB (hnRNP AB), and to investigate their clinical significance in hepatocellular carcinoma (HCC). Methods: We carried out mass spectrometry to reveal the specific peptides of KRAB-associated protein 1 (Kap1) and hnRNPAB, and verified their interaction by immunocoprecipitation and western blotting. Expression of hnRNPAB/Kap1 proteins were detected by immunohistochemical staining in the tissue microarrays. Categorical data were analyzed by the chi square test or Fisher exact test; enumeration data between groups were compared using Student t-test or Wilcocon signed rank test; the cumulative recurrence and survival rates were evaluated using the Kaplan-Meier method and the differences were assessed using the log-rank test. Results: We identified Kap1 as a molecular partner for hnRNPAB in HCCLM3 cells and HepG2 cells as well. We found that the 5-year survival rate of the Kap1high patients was significantly lower than the survival rate of those of the Kap1low group (36% vs 59% , HR = 1.67, P < 0.001). Similarly, Kap1high HCC patients had the poorest prognosis at 5-years, with higher cumulative recurrence rate than Kap1low patients (72% vs 54%, HR = 1.66, P = 0.001). Univariate and Multivariate analyses revealed that hnRNPAB /Kap1 alone (HR = 1.35 /1.28, P = 0.001) or in combination with Kap1 (HR =1.24 /1.27, P < 0.05) were independent prognostic indicators for overall survival and time to recurrence. Conclusion: In HCC cells, hnRNPAB and Kap1 form protein complexes. The expression levels of hnRNPAB alone or in combination with Kap1 in HCC patients are important because they provide not only a predictor for HCC prognosis but also a therapeutic target for future studies.

Odorant Sensory Input Modulates DNA Secondary Structure Formation and Heterogeneous Ribonucleoprotein Recruitment on the Tyrosine Hydroxylase and Glutamic Acid Decarboxylase 1 Promoters in the Olfactory Bulb.

Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. For a subset of olfactory bulb interneurons, activity-dependent changes in GABA are reflected by corresponding changes in Glutamate decarboxylase 1 (Gad1) expression levels. Mechanisms regulating Gad1 promoter activity are poorly understood, but here we show that a conserved G:C-rich region in the mouse Gad1 proximal promoter region both recruits heterogeneous nuclear ribonucleoproteins (hnRNPs) that facilitate transcription and forms single-stranded DNA secondary structures associated with transcriptional repression. This promoter architecture and function is shared with Tyrosine hydroxylase (Th), which is also modulated by odorant-dependent activity in the olfactory bulb. This study shows that the balance between DNA secondary structure formation and hnRNP binding on the mouse Th and Gad1 promoters in the olfactory bulb is responsive to changes in odorant-dependent sensory input. These findings reveal that Th and Gad1 share a novel transcription regulatory mechanism that facilitates sensory input-dependent regulation of dopamine and GABA expression.SIGNIFICANCE STATEMENT Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. This study shows that transcription of genes encoding rate-limiting enzymes for GABA and dopamine biosynthesis (Gad1 and Th, respectively) in the mammalian olfactory bulb is regulated by G:C-rich regions that both recruit heterogeneous nuclear ribonucleoproteins (hnRNPs) to facilitate transcription and form single-stranded DNA secondary structures associated with repression. hnRNP binding and formation of DNA secondary structure on the Th and Gad1 promoters are mutually exclusive, and odorant sensory input levels regulate the balance between these regulatory features. These findings reveal that Th and Gad1 share a transcription regulatory mechanism that facilitates odorant-dependent regulation of dopamine and GABA expression levels.