Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.     

The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation

hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.     

Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta. Evidence favoring identity with heterogeneous nuclear ribonucleoprotein E1

The interaction of an 18-base cis-element in the 5'-untranslated region of human folate receptor (FR)-alpha mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539-25547). This trans-factor was isolated to apparent homogeneity as a 43- and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA cis-element in the 3'-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as alphaCP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.     

Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This 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 them sensitive to rapamycin-induced G(1) 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) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting 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 Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.     

Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.     

HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.     

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.     

HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition

Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.     

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.     

c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs

hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.     

Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes

TOP mRNAs (contain a 5' terminal oligopyrimidine tract) are differentially translated in rapamycin-treated human B lymphocytes. Following rapamycin treatment, ribosomal protein (rp) and translation elongation factor TOP mRNAs were translationally repressed, whereas hnRNP A1 TOP mRNA was not. Poly(A)-binding protein (Pabp1) TOP mRNA was translationally repressed under all conditions tested. To investigate the mechanism involved, chimeric mRNAs containing the hnRNP A1 5' untranslated region (UTR) linked to the human growth hormone (hGH) reporter were analyzed. Wild-type hnRNP A1 construct mRNA behaved similarly to endogenous hnRNP A1, whereas a single mutation (guanosine to cytidine) within the TOP element resulted in increased translational regulation. These results suggest that TOP mRNA translation can be modulated and that all TOP mRNAs are not translated with equal efficiency.