Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1).

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Prognostic Value of GDF-15 in Predicting Prolonged Intensive Care Stay following Cardiac Surgery: A Pilot Study.

INTRODUCTION: Predicting intensive care unit length of stay and outcome following cardiac surgery is currently based on clinical parameters. Novel biomarkers could be employed to improve the prediction models. MATERIALS AND METHODS: We performed a qualitative cytokine screening array to identify highly expressed biomarkers in preoperative blood samples of cardiac surgery patients. After identification of one highly expressed biomarker, growth differentiation factor 15 (GDF-15), a quantitative ELISA was undertaken. Preoperative levels of GDF-15 were compared in regard to duration of intensive care stay, cardiopulmonary bypass time, and indicators of organ dysfunction. RESULTS: Preoperatively, GDF-15 was highly expressed in addition to several less highly expressed other biomarkers. After qualitative analysis, we could show that preoperatively raised levels of GDF-15 were positively associated with prolonged ICU stay exceeding 48 h (median 713 versus 1041 pg/ml, p = 0.003). It was also associated with prolonged mechanical ventilation and rates of severe sepsis but not with dialysis rates or cardiopulmonary bypass time. In univariate regression, raised GDF-15 levels were predictive of a prolonged ICU stay (OR 1.01, 95% confidence interval 1-1.02, and p = 0.029). On ROC curves, GDF-15 was found to predict prolonged ICU stay (AUC = 0.86, 95% confidence interval 0.71-0.99, and p = 0.003). CONCLUSION: GDF-15 showed potential as predictor of prolonged intensive care stay following cardiac surgery, which might be valuable for risk stratification models.

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.

P23 Acts as Functional RBP in the Macrophage Inflammation Response.

Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.

Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.

Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

RNA-Binding Proteins in the Control of LPS-Induced Macrophage Response.

Innate immune response is triggered by pathogen components, like lipopolysaccharides (LPS) of gram-negative bacteria. LPS initiates Toll-like receptor 4 (TLR4) signaling, which involves mitogen activated protein kinases (MAPK) and nuclear factor kappa B (NFκB) in different pathway branches and ultimately induces inflammatory cytokine and chemokine expression, macrophage migration and phagocytosis. Timely gene transcription and post-transcriptional control of gene expression confer the adequate synthesis of signaling molecules. As trans-acting factors RNA binding proteins (RBPs) contribute significantly to the surveillance of gene expression. RBPs are involved in the regulation of mRNA processing, localization, stability and translation. Thereby they enable rapid cellular responses to inflammatory mediators and facilitate a coordinated systemic immune response. Specific RBP binding to conserved sequence motifs in their target mRNAs is mediated by RNA binding domains, like Zink-finger domains, RNA recognition motifs (RRM), and hnRNP K homology domains (KH), often arranged in modular arrays. In this review, we focus on RBPs Tristetraprolin (TTP), human antigen R (HUR), T-cell intracellular antigen 1 related protein (TIAR), and heterogeneous ribonuclear protein K (hnRNP K) in LPS induced macrophages as primary responding immune cells. We discuss recent experiments employing RNA immunoprecipitation and microarray analysis (RIP-Chip) and newly developed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), photoactivatable ribonucleoside-enhanced crosslinking (PAR-iCLIP) and RNA sequencing techniques (RNA-Seq). The global mRNA interaction profile analysis of TTP, HUR, TIAR, and hnRNP K exhibited valuable information about the post-transcriptional control of inflammation related gene expression with a broad impact on intracellular signaling and temporal cytokine expression.

Characterization of acute myeloid leukemia with del(9q) - Impact of the genes in the minimally deleted region.

Acute myeloid leukemia is an aggressive disease that arises from clonal expansion of malignant hematopoietic precursor cells of the bone marrow. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of acute myeloid leukemia patients. Our deletion analysis in a cohort of 31 del(9q) acute myeloid leukemia patients further supports the importance of a minimally deleted region composed of seven genes potentially involved in leukemogenesis: GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2. Importantly, among them HNRNPK, encoding heterogeneous nuclear ribonucleoprotein K is proposed to function in leukemogenesis. We show that expression of HNRNPK and the other genes of the minimally deleted region is significantly reduced in patients with del(9q) compared with normal karyotype acute myeloid leukemia. Also, two mRNAs interacting with heterogeneous nuclear ribonucleoprotein K, namely CDKN1A and CEBPA are significantly downregulated. While the deletion size is not correlated with outcome, associated genetic aberrations are important. Patients with an additional t(8;21) show a good prognosis. RUNX1-RUNX1T1, which emerges from the t(8;21) leads to transcriptional down-regulation of CEBPA. Acute myeloid leukemia patients with mutations in CEBPA have a good prognosis as well. Interestingly, in del(9q) patients with CEBPA mutation mRNA levels of HNRNPK and the other genes located in the minimally deleted region is restored to normal karyotype level. Our data indicate that a link between CEBPA and the genes of the minimally deleted region, among them HNRNPK contributes to leukemogenesis in acute myeloid leukemia with del(9q).

Identification of RNA-binding Proteins in Macrophages by Interactome Capture.

Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.

Translational control mediated by hnRNP K links NMHC IIA to erythroid enucleation.

Post-transcriptional regulation is crucial for structural and functional alterations in erythropoiesis. Enucleation of erythroid progenitors precedes reticulocyte release into circulation. In enucleated cells, reticulocyte 15-lipoxygenase (r15-LOX, also known as ALOX15) initiates mitochondria degradation. Regulation of r15-LOX mRNA translation by hnRNP K determines timely r15-LOX synthesis in terminal maturation. K562 cells induced for erythroid maturation recapitulate enucleation and mitochondria degradation. HnRNP K depletion from maturing K562 cells results in enhanced enucleation, which even occurs independently of maturation. We performed RIP-Chip analysis to identify hnRNP K-interacting RNAs comprehensively. Non-muscle myosin heavy chain (NMHC) IIA (also known as MYH9) mRNA co-purified with hnRNP K from non-induced K562 cells, but not from mature cells. NMHC IIA protein increase in erythroid maturation at constant NMHC IIA mRNA levels indicates post-transcriptional regulation. We demonstrate that binding of hnRNP K KH domain 3 to a specific sequence element in the NMHC IIA mRNA 3'UTR mediates translation regulation in vitro Importantly, elevated NMHC IIA expression results in erythroid-maturation-independent enucleation as shown for hnRNP K depletion. Our data provide evidence that hnRNP-K-mediated regulation of NMHC IIA mRNA translation contributes to the control of enucleation in erythropoiesis.

miR-155 targets Caspase-3 mRNA in activated macrophages.

To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids.

Abstract Analysis of arginine methylation, which affects specific protein interactions in eukaryotic cells, requires access to methylated protein for biophysical and biochemical studies. Methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon co-expression with protein arginine methyltransferase 1 in E. coli was monitored by mass spectrometry and found to be identical to the modification of hnRNP K purified from mammalian cells. Recombinant non-methylated and arginine-methylated hnRNP K (MethnRNP K) were used to characterize self-aggregation and nucleic acid binding. Analytical ultracentrifugation and static light scattering experiments revealed that hnRNP K methylation does not impact reversible self-aggregation, which can be prevented by high ionic strength and organic additives. Filter binding assays were used to compare the binding of non-methylated and MethnRNP K to the pyrimidine repeat-containing differentiation control element (DICE) of reticulocyte 15-lipoxygenase mRNA 3' UTR. No affinity differences were detected for both hnRNP K variants. A series of oligonucleotides carrying various numbers of C4 motifs at different positions was used in steady state competition assays with fluorescently-labeled functional differentiation control element (2R). Quantitative evaluation indicated that all hnRNP K homology domains of hnRNP K contribute differentially to RNA binding, with KH1-KH2 acting as a tandem domain and KH3 as an individual binding domain.

DDX6 and its orthologs as modulators of cellular and viral RNA expression.

DDX6 (Rck/p54), a member of the DEAD-box family of helicases, is highly conserved from unicellular eukaryotes to vertebrates. Functions of DDX6 and its orthologs in dynamic ribonucleoproteins contribute to global and transcript-specific messenger RNA (mRNA) storage, translational repression, and decay during development and differentiation in the germline and somatic cells. Its role in pathways that promote mRNA-specific alternative translation initiation has been shown to be linked to cellular homeostasis, deregulated tissue development, and the control of gene expression in RNA viruses. Recently, DDX6 was found to participate in mRNA regulation mediated by miRNA-mediated silencing. DDX6 and its orthologs have versatile functions in mRNA metabolism, which characterize them as important post-transcriptional regulators of gene expression.

Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced macrophage activation.

Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signaling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous nuclear ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating hematopoietic cells, was studied in noninduced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed several mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor-ß-activated kinase 1 (TAK1) a central player in TLR4 signaling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis or stability but enhances TAK1 mRNA translation, resulting in elevated TNF-α, IL-1ß, and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in noninduced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesized TAK1 boosts macrophage inflammatory response.

Identification of DEAD-box RNA helicase 6 (DDX6) as a cellular modulator of vascular endothelial growth factor expression under hypoxia.

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5'-untranslated region (5'-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5'-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5'-UTR and 3'-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5'-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.

Binding of the heterogeneous ribonucleoprotein K (hnRNP K) to the Epstein-Barr virus nuclear antigen 2 (EBNA2) enhances viral LMP2A expression.

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.

Precision mechanics with multifunctional tools: how hnRNP K and hnRNPs E1/E2 contribute to post-transcriptional control of gene expression in hematopoiesis.

Studies on the post-transcriptional control of gene expression in hematopoietic cells have uncovered that a subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs) is involved in cytoplasmic gene regulation. Among them hnRNP K and hnRNPs E1/E2 share common structural motifs, the hnRNP K homology (KH) domains that provide a functional basis for RNA binding. Specific sub-cellular localization and differentiation dependent post-translational modifications modulate the interaction of these proteins with mRNA and proteins in messenger ribonucleoprotein complexes (mRNPs), the latter generating connectivity to cell signaling events. As components of different mRNPs, hnRNP K and hnRNPs E1/E2 function as crucial modulators of mRNA stability and translation in hematopoietic cell differentiation.

DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules.

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I.

Mammalian cleavage factor I (CF I(m)) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I(m)25, CF I(m)59, CF I(m)68). It is part of the cleavage and polyadenylation complex responsible for processing the 3' ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3' processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I(m)68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I(m)68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I(m)68 we could exclusively identify arginines in a GGRGRGRF or "GAR" motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I(m)68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I(m)59 and CF I(m)68. The results suggest that native-as compared with recombinant-protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I(m) methylation in the context of RNA export is discussed.

Plakophilin 1 stimulates translation by promoting eIF4A1 activity.

Plakophilins 1-3 (PKP1-3) are desmosomal proteins of the p120(ctn) family of armadillo-related proteins that are essential for organizing the desmosomal plaque. Recent findings identified PKPs in stress granules, suggesting an association with the translational machinery. However, a role of PKPs in controlling translation remained elusive so far. In this study, we show a direct association of PKP1 with the eukaryotic translation initiation factor 4A1 (eIF4A1). PKP1 stimulated eIF4A1-dependent translation via messenger RNA cap and encephalomyocarditis virus internal ribosomal entry site (IRES) structures, whereas eIF4A1-independent translation via hepatitis C virus IRES was not affected. PKP1 copurified with eIF4A1 in the cap complex, and its overexpression stimulated eIF4A1 recruitment into cap-binding complexes. At the molecular level, PKP1 directly promoted eIF4A1 adenosine triphosphatase activity. The stimulation of translation upon PKP1 overexpression correlated with the up-regulation of proliferation and cell size. In conclusion, these findings identify PKP1 as a regulator of translation and proliferation via modulation of eIF4A1 activity and suggest that PKP1 controls cell growth in physiological and pathological conditions.