hnRNP A1 Protein Research Articles

Thermodynamic and Phylogenetic Insights into hnRNP A1 Recognition of the HIV-1 Exon Splicing Silencer 3 Element

Complete expression of the HIV-1 genome requires balanced usage of suboptimal splice sites. The 3′ acceptor site A7 (ssA7) is negatively regulated in part by an interaction between the host hnRNP A1 protein and a viral splicing silencer (ESS3). Binding of hnRNP A1 to ESS3 and other upstream silencers is sufficient to occlude spliceosome assembly. Efforts to understand the splicing repressive properties of hnRNP A1 on ssA7 have revealed hnRNP A1 binds specific sites within the context of a highly folded RNA structure; however, biochemical models assert hnRNP A1 disrupts RNA structure through cooperative spreading. In an effort to improve our understanding of the ssA7 binding properties of hnRNP A1, herein we have performed a combined phylogenetic and biophysical study of the interaction of its UP1 domain with ESS3. Phylogenetic analyses of group M sequences (x̅ = 2860) taken from the Los Alamos HIV database reveal the ESS3 stem loop (SL3ESS3) structure has been conserved throughout HIV-1 evolution, despite variations in primary sequence. Calorimetric titrations with UP1 clearly show the SL3ESS3 structure is a critical binding determinant because deletion of the base-paired region reduces the affinity by ∼150-fold (Kd values of 27.8 nM and 4.2 μM). Cytosine substitutions of conserved apical loop nucleobases show UP1 preferentially binds purines over pyrimidines, where site-specific interactions were detected via saturation transfer difference nuclear magnetic resonance. Chemical shift mapping of the UP1–SL3ESS3 interface by 1H–15N heteronuclear single-quantum coherence spectroscopy titrations reveals a broad interaction surface on UP1 that encompasses both RRM domains and the inter-RRM linker. Collectively, our results describe a UP1 binding mechanism that is likely different from current models used to explain the alternative splicing properties of hnRNP A1.

HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

A synonymous polymorphic variation in ACADM exon 11 affects splicing efficiency and may affect fatty acid oxidation

In recent studies combining genome-wide association and tandem-MS based metabolic profiling, a single-nucleotide polymorphism (SNP), rs211718C>T, located far upstream of the MCAD gene (ACADM) was found to be associated with serum concentrations of medium-chain acylcarnitines indicating improved beta-oxidation of medium-chain fatty acids. We examined the functional basis for this association and identified linkage between rs211718 and the intragenic synonymous polymorphic variant c.1161A>G in ACADM exon 11 (rs1061337). Employing minigene studies we show that the c.1161A allele is associated with exon 11 missplicing, and that the c.1161G allele corrects this missplicing. This may result in production of more full length MCAD protein from the c.1161G allele. Our analysis suggests that the improved splicing of the c.1161G allele is due to changes in the relative binding of splicing regulatory proteins SRSF1 and hnRNP A1. Using publicly available pre-aligned RNA-seq data, we find that the ACADM c.1161G allele is expressed at significantly higher levels than the c.1161A allele across different tissues. This supports that c.1161A>G is a functional SNP, which leads to higher MCAD expression, perhaps due to improved splicing.This study is a proof of principle that synonymous SNPs are not neutral. By changing the binding sites for splicing regulatory proteins they can have significant effects on pre-mRNA splicing and thus protein function. In addition, this study shows that for a sequence variation to have an effect, it might need to change the balance in the relative binding of positive and negative splicing factors.

Functional Involvements of Heterogeneous Nuclear Ribonucleoprotein A1 in Smooth Muscle Differentiation from Stem Cells In Vitro and In Vivo

To investigate the functional involvements of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in smooth muscle cell (SMC) differentiation from stem cells, embryonic stem cells were cultivated on collagen IV-coated plates to allow for SMC differentiation. We found that hnRNPA1 gene and protein expression was upregulated significantly during differentiation and coexpressed with SMC differentiation markers in the stem cell-derived SMCs as well as embryonic SMCs of 12.5 days of mouse embryos. hnRNPA1 knockdown resulted in downregulation of smooth muscle markers and transcription factors, while enforced expression of hnRNPA1 enhanced the expression of these genes. Importantly, knockdown of hnRNPA1 also resulted in impairment of SMC differentiation in vivo. Moreover, we demonstrated that hnRNPA1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Acta2 and Tagln genes using luciferase and chromatin immunoprecipitation assays. We further demonstrated that the binding sites for serum response factor (SRF), a well-investigated SMC transcription factor, within the promoter region of the Acta2 and Tagln genes were responsible for hnRNPA1-mediated Acta2 and Tagln gene expression using in vitro site-specific mutagenesis and luciferase activity analyses. Finally, we also demonstrated that hnRNPA1 upregulated the expression of SRF, myocyte-specific enhancer factor 2c (MEF2c), and myocardin through transcriptional activation and direct binding to promoters of the SRF, MEF2c, and Myocd genes. Our findings demonstrated that hnRNPA1 plays a functional role in SMC differentiation from stem cells in vitro and in vivo. This indicates that hnRNPA1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine.

Antibodies to an Intracellular Antigen Penetrate Neuronal Cells and Cause Deleterious Effects

Multiple sclerosis (MS) is an autoimmune disease that is increasingly being recognized as a neurodegenerative disorder. Patients with MS produce autoantibodies to heterogenous nuclear ribonucleoprotein A1 (hnRNPA1). A multitude of studies indicate that T-lymphocytes, B-lymphocytes and macrophages contribute to MS pathogenesis. However, a direct autoantibody impact on neuronal cells has received limited attention. This could be explained by the general belief that autoantibodies lack the ability to penetrate neurons. hnRNP A1 is an intracellular RNA binding protein that exports RNA from the nucleus to the cytoplasm. In this study, we investigated possible mechanisms of antibody penetration into neuronal cells. Our results show that anti-hnRNP A1 antibodies and control IgG penetrate SK-N-SH neuronal cells through clathrin-mediated endocytosis. In contrast to control antibodies, anti-hnRNP A1 antibodies produced deleterious effects on the neuronal cells including altered ATP levels and increased caspase 3/7 levels (leading to apoptosis). Remarkably, anti-hnRNP A1 antibodies that targeted the hnRNP A1 M9 domain (its nuclear export/localization sequence) caused redistribution of endogenous hnRNPA1 protein in neuronal cells. These findings indicate that anti-hnRNPA1 antibodies might contribute to the pathogenesis of MS.

Anti-Leukemic Activity of the CRM1 Inhibitor KPT-330 in Advanced CML and Ph+ ALL

AbstractAbstract 35As tyrosine kinase inhibitors (TKIs) do not induce long-term response in blast crisis chronic myeloid leukemia (CML-BC) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and are unable to kill quiescent Ph+ hematopoietic stem cells, alternative therapies targeting dysregulated pathways in BCR-ABL1+ acute leukemias are needed. CRM1, a karyopherin aberrantly overexpressed in several cancers, controls the nuclear export of proteins (e.g. ABL1, SET, p53, p21, FOXO and RB) that regulate normal and malignant hematopoietic cell survival, self-renewal and proliferation. Here we show that enhanced CRM1 expression also occurs in Ph+ leukemias and the clinical stage small molecule inhibitor of CRM1, KPT-330, has a detrimental effect on malignant but not normal hematopoietic progenitors.Specifically, a substantial upregulation (≥ 65% increase) in the expression levels of CRM1 was detected by immunoblot in CD34+ progenitors from bone marrow (BM) of leukemic primary samples (n=3), when compared to CD34+ progenitors from healthy (n=3) donors. Interestingly, CRM1 protein levels in CML CD34+ cells were markedly but not totally reduced by treatment with the ABL1 kinase inhibitor Imatinib (1μM, 72h), suggesting that BCR-ABL1-driven pathways are not the only factor contributing to CRM1 upregulation. Additionally, treatment of primary CML cells with KPT-330 (1μM, 72h) resulted in a 75% reduction in BCR-ABL1 kinase activity, consistent with an interrelationship between BCR-ABL1 and CRM1 activities. Finally, the ability of BCR-ABL1 to induce CRM1 expression was demonstrated upon ectopic BCR-ABL1 expression in myeloid 32Dcl3 precursor cells in which CRM1 protein levels became ∼10-fold higher.To determine the therapeutic relevance of CRM1 inhibition, cell survival was assessed in MACS-purified CD34+ progenitors. Treatment with KPT-330 resulted in ≥80% induction of apoptosis in Ph+ (n=5) CML (2μM, 72hr) and B-ALL (500nM, 72h) CD34+ cells. Conversely, KPT-330 exerted only a minimal effect on the survival of CD34+ progenitors from healthy donor BM (15–30% Annexin V positive, KPT-330 0.5–2μM, 72h). Consistent with the existence of BCR-ABL1-independent signaling leading to increased CRM1 expression, marked apoptosis was also observed in KPT-330-treated CD34+ progenitors isolated from a Ph-negative B-ALL patient. Thus, CRM1 inhibitor-based therapies might also benefit BCR-ABL1-negative leukemias. To formally determine the effects of KPT-330 on leukemic cell survival, methylcellulose-based clonogenic assays were performed. KPT-330 treatment (1μM) resulted in almost total suppression (97% reduction) of the clonogenic potential of leukemic but not normal (30% reduction) CD34+ progenitors.Because of evidence that CRM1 directly interacts with the protein phosphatase 2A (PP2A) inhibitor SET, and that CRM1 inhibition alters trafficking of hnRNP A1, a direct regulator of the SET-PP2A interplay in Ph+ leukemias, it is conceivable that the anti-leukemic activity of KPT-330 is, at least in part, mediated by PP2A activation. Indeed, KPT-330 treatment (1μM, 48hr) of BCR-ABL1+ cell lines resulted in full restoration of PP2A activity, comparable to levels observed in BCR-ABL1-negative cells. Accordingly, KPT-330 treatment (1μM, 12h) of 32Dcl3BCR-ABL1 cells caused a nuclear accumulation (4-fold increase) and cytoplasmic decrease (95% lower) of SET protein levels, as indicated by both confocal microscopy and immunoblots of subcellular fractions. Moreover, KPT-330-treated cells showed altered hnRNP A1 cellular distribution and overall downregulation. However, unexpectedly, hnRNP A1 protein levels were proportionally higher in the cytoplasm and lower in the nucleus, suggesting that the deleterious effect of CRM1 inhibition on hnRNP A1 might not depend on direct inhibition of hnRNP-A1 nuclear export.Because KPT-330 displays favorable ADME properties and has been shown to alleviate leukemic burden using in vivo models of different cancers, we are currently testing the anti-leukemic activity of this compound in a mouse model of CML-BC. We do expect a profound inhibition of leukemogenesis, and all treated mice are currently alive with no signs of toxicity. Thus, CRM1 inhibition by KPT-330 represents a potential new therapeutic avenue which could easily be exploited in malignancies like CML-BC and Ph+ ALL that show a dismal outcome to current available therapies. Disclosures:Walker:Karyopharm Therapeutics Inc: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Perrotti:Karyopharm Therapeutics Inc: Research Funding.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

Abstract 2946: Post-transcriptional regulators of microRNA biogenesis regulate pathogenesis of cancer

Abstract MicroRNAs (miRs) have emerged as key regulators of both normal and pathologic phenotypes, including cancer. Fine grained regulation of their biogenesis, however, is still poorly understood and only a few of their key regulators have been characterized. In order to understand the extent and specificity of miR-biogenesis control, as well as the role of miR-biogenesis regulators in tumorigenesis and cancer progression, we set out to identify these regulators and profile their targets. We developed a genome-wide computational screening method that leverages miR and gene expression profiles in hundreds of samples to identify potential biogenesis regulators in glioma and ovarian cancer. In total, miRage predicted thirty miR-biogenesis regulators that are common to glioma and ovarian cancer tumors, and hundreds of glioma-specific and ovarian-cancer-specific regulators. Predicted regulators include known biogenesis regulators DGCR8, HNRNPA1, DDX5, LIN28 and SMAD family proteins. We validated selected regulators that are predicted to regulate miRs in both cancers and are differentially expressed across glioma prognosis. Differential expression of (1) mature miRs, (2) their host genes, (3) co-transcribed miRs, (4) miR precursors, and (5) pri-miRs were measured upon ectopic expression or RNAi-mediated silencing of the predicted regulators in a glioma cell line. DDX10 was predicted to up-regulate miR-218 biogenesis, and we showed that its ectopic expression up-regulates miR-218 at the pri-miRNA stage; up-regulation of DDX10 may account for miR-218 over-expression in poor-prognosis tumors. High-throughput screening by nanostring nCounter and fluidigm systems suggest that DDX10 regulation is broader effect than regulation by the known biogenesis regulator, DDX17. In total, we report on nearly a dozen validated miR-biogenesis regulators that were predicted by our computational screens. Our results suggest that miR biogenesis is a complex, context specific, and finely-regulated process, and that miR biogenesis regulators may influence tumor initiation and progression by altering the expression of specific tumor-suppressor and oncomirs or by modifying large miR programs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2946. doi:1538-7445.AM2012-2946

Concentration-dependent control of pyruvate kinase M mutually exclusive splicing by hnRNP proteins

Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB have a crucial role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as those found in most cancer cells, hnRNPA1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites, such that exon 9 is included and exon 10 mainly excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together, our results provide a mechanism by which a few general factors control alternative splicing of a widely expressed transcript.

MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a KD value of 82.7nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (KD: 260nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5–50μM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

A leaky splicing mutation affecting SMN1 exon 7 inclusion explains an unexpected mild case of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting, in most cases, from homozygous deletions of the SMN1 gene or, in rare cases, from SMN1 intragenic mutations. Here we describe the identification and characterization of c.835-3C>T, a novel SMA-causing mutation detected in the intron 6 of the single SMN1 allele of a type IV SMA patient. We demonstrate both ex vivo and in vivo that c.835-3C>T is a deleterious splicing mutation that induces a modest but unequivocal exclusion of exon 7 from the SMN1 transcripts, its "leakiness" explaining the exceptionally mild phenotype of this patient. This mutation creates a putative high-affinity binding site for the splicing repressor protein hnRNP A1 overlapping the splice acceptor site of exon 7 (UAG|GGU). Our findings support the current therapeutic strategies aiming at correcting exon 7 splicing in SMA patients, and bring clues about the level of exon 7 inclusion required to achieve a therapeutic effect.

Prediction of single‐nucleotide substitutions that result in exon skipping: identification of a splicing silencer inBRCA1exon 6

Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among disease-causing exonic substitutions is unknown. Here, we have tested missense and silent mutations deposited in the BRCA1 mutation databases of unclassified variants for their effects on exon inclusion. Analysis of 21 BRCA1 variants using minigene assays revealed a single exon-skipping mutation c.231G>T. Comprehensive mutagenesis of an adjacent 12-nt segment showed that this silent mutation resulted in a higher level of exon skipping than the 35 other single-nucleotide substitutions. Exon inclusion levels of mutant constructs correlated significantly with predicted splicing enhancers/silencers, prompting the development of two online utilities freely available at http://www.dbass.org.uk. EX-SKIP quickly estimates which allele is more susceptible to exon skipping, whereas HOT-SKIP examines all possible mutations at each exon position and identifies candidate exon-skipping positions/substitutions. We demonstrate that the distribution of exon-skipping and disease-associated substitutions previously identified in coding regions was biased toward top-ranking HOT-SKIP mutations. Finally, we show that proteins 9G8, SC35, SF2/ASF, Tra2, and hnRNP A1 were associated with significant alterations of BRCA1 exon 6 inclusion in the mRNA. Together, these results facilitate prediction of exonic substitutions that reduce exon inclusion in mature transcripts.

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.

IL-6-induced Enhancement of c-Myc Translation in Multiple Myeloma Cells: CRITICAL ROLE OF CYTOPLASMIC LOCALIZATION OF THE RNA-BINDING PROTEIN hnRNP A1

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5'-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.

Turning on a Fuel Switch of Cancer: hnRNP Proteins Regulate Alternative Splicing of Pyruvate Kinase mRNA

Unlike normal cells, which metabolize glucose by oxidative phosphorylation for efficient energy production, tumor cells preferentially metabolize glucose by aerobic glycolysis, which produces less energy but facilitates the incorporation of more glycolytic metabolites into the biomass needed for rapid proliferation. The metabolic shift from oxidative phosphorylation to aerobic glycolysis is partly achieved by a switch in the splice isoforms of the glycolytic enzyme pyruvate kinase. Although normal cells express the pyruvate kinase M1 isoform (PKM1), tumor cells predominantly express the M2 isoform (PKM2). Switching from PKM1 to PKM2 promotes aerobic glycolysis and provides a selective advantage for tumor formation. The PKM1/M2 isoforms are generated through alternative splicing of two mutually exclusive exons. A recent study shows that the alternative splicing event is controlled by heterogeneous nuclear ribonucleoprotein (hnRNP) family members hnRNPA1, hnRNPA2, and polypyrimidine tract binding protein (PTB; also known as hnRNPI). These findings not only provide additional evidence that alternative splicing plays an important role in tumorigenesis, but also shed light on the molecular mechanism by which hnRNP proteins regulate cell proliferation in cancer.

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions.Methodology/Principal FindingsUsing real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1.Conclusions/SignificanceWe found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.

Quaking I controls a unique cytoplasmic pathway that regulates alternative splicing of myelin-associated glycoprotein

Precise control of alternative splicing governs oligodendrocyte (OL) differentiation and myelination in the central nervous system (CNS). A well-known example is the developmentally regulated expression of splice variants encoding myelin-associated glycoprotein (MAG), which generates two protein isoforms that associate with distinct cellular components crucial for axon-glial recognition during myelinogenesis and axon-myelin stability. In the quakingviable (qk(v)) hypomyelination mutant mouse, diminished expression of isoforms of the selective RNA-binding protein quaking I (QKI) leads to severe dysregulation of MAG splicing. The nuclear isoform QKI-5 was previously shown to bind an intronic element of MAG and modulate alternative exon inclusion from a MAG minigene reporter. Thus, QKI-5 deficiency was thought to underlie the defects of MAG splicing in the qk(v) mutant. Surprisingly, we found that transgenic expression of the cytoplasmic isoform QKI-6 in the qk(v) OLs completely rescues the dysregulation of MAG splicing without increasing expression or nuclear abundance of QKI-5. In addition, cytoplasmic QKI-6 selectively associates with the mRNA that encodes heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a well-characterized splicing factor. Furthermore, QKI deficiency in the qk(v) mutant results in abnormally enhanced hnRNPA1 translation and overproduction of the hnRNPA1 protein but not hnRNPA1 mRNA, which can be successfully rescued by the QKI-6 transgene. Finally, we show that hnRNPA1 binds MAG pre-mRNA and modulates alternative inclusion of MAG exons. Together, these results reveal a unique cytoplasmic pathway in which QKI-6 controls translation of the splicing factor hnRNPA1 to govern alternative splicing in CNS myelination.

Abstract 1595: Phosphomimetic mutation of hnRNP A1 inhibits AKT-dependent IRES function and results in mTOR inhibitor sensitivity

Abstract The relative activity of the Akt kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mTOR inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein hnRNP A1 regulates a salvage pathway which facilitates IRES-dependent mRNA translation of critical cellular determinants in an Akt-dependent manner following mTOR inhibitor exposure. This salvage pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent Akt resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated Akt activity rendering cells sensitive to rapamycin induced G1 arrest as a result of the inhibition of global eIF-4E-mediated translation. Akt phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) which binds both the cyclin D1 and c-MYC IRES RNAs in vitro and results in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which Akt is conditionally active, we demonstrate that overexpression of this mutant renders quiescent Akt-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated Akt is strongly correlated with elevated phospho- ser199-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 ser199 regulates the Akt-dependent sensitivity of cells to rapamycin and may have utility as a surrogate biomarker to stratify those tumors which may be most sensitive to mTOR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1595.