Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3' untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3' untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site-mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.

Fragile X syndrome is a common form of inherited mental retardation. Most fragile X patients exhibit mutations in the fragile X mental retardation gene 1 (FMR1) that lead to transcriptional silencing and hence to the absence of the fragile X mental retardation protein (FMRP). Since FMRP is an RNA-binding protein which associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. In the present study, we show that FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologues of FMRP, FXR1 and FXR2, failed to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins FXR1 and FXR2. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. Our data support the notion that inhibition of translation may be a function of FMRP in vivo. We further suggest that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome.

hnRNP K and hnRNP E1/E2 regulate human papilloma virus type 16 (HPV-16) L2 capsid protein mRNA and reticulocyte 15-lipoxygenase (LOX) mRNA expression in the course of cellular differentiation. The expression of the virus capsid protein L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium by repression of L2 mRNA translation in the deeper layers (13). The underlying inhibitory mechanism employs hnRNPs K and E1/E2 interacting with a specific cis-acting element in the 3Ј end of L2 mRNA (5). LOX is a key enzyme in erythroid cell differentiation. It can attack phospholipids of the mitochondrial membranes and participates in their breakdown in late reticulocytes (20,24). LOX expression is temporally restricted by translational silencing of LOX mRNA in erythroid precursor cells in the bone marrow and in early reticulocytes (12). The differentiation control element (DICE) in the 3Ј untranslated region (UTR) of LOX mRNA binds the KH domain proteins hnRNP K and hnRNP E1 to form translationally silenced mRNPs in immature erythroid cells (15,16). The hnRNP K/E1-DICE complex blocks 80S ribosome assembly by inhibition of 60S ribosomal subunit joining (15). This erythroid silencing mechanism can be recapitulated in vitro and in HeLa cells transfected with DICE-regulated reporter mRNAs and hnRNP K alone or together with hnRNP E1 (16). Like LOX mRNA silencing, the HPV-16 L2 mRNA mechanism has been shown to operate in HeLa cells as well (5).The C-terminal part of hnRNP K contains proline-rich domains, which enable hnRNP K to interact with the SH3 domains of members of the Src kinase family (3) such as c-Src itself (23,25,27), Fyn, and Lyn (27). c-Src and Lck, a further member of the Src kinase family, have been shown to be able to phosphorylate hnRNP K in vitro and to affect its binding to RNA (19). The functional significance of hnRNP K tyrosine phosphorylation by members of the Src family of kinases is as yet unknown.Here we show that hnRNP K specifically binds and activates c-Src. c-Src mediates tyrosine phosphorylation of hnRNP K and inhibition of its DICE binding activity. Moreover, we demonstrate that c-Src kinase specifically regulates hnRNP K function as a translational silencer in vivo. Our results identify a specific role of c-Src in posttranscriptional regulation via hnRNP K, and suggest a mechanism for how the differentiation-dependent translation of cellular and viral RNA could be activated in maturing cells. MATERIALS AND METHODSPlasmids. For luciferase (LUC) indicator constructs, the LUC cDNA from pGEM-LUC (Promega) was inserted into pSG5 (16). LUC-DICE and LUC-NR were made by insertion of the DICE or nonregulatory (NR) sequences of the LOX mRNA 3Ј UTR into the EcoRV site of pSG5 before insertion of the LUC open reading frame (18). pSG5-His-hnRNP K was generated using pSG5-hnRNP K (16) by insertion of an oligonucleotide coding for 10 histidine residues between the SmaI and XhoI sites, N-terminal to hnRNP K. The tyrosine-tophenylalanine mutants pSG5-His-...

Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 59 and 39 termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein.HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 59-39 interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.

During red blood cell differentiation, the mRNA encoding rabbit erythroid 15‐lipoxygenase (LOX) is synthesized in the early stages of erythropoiesis, but is only activated for translation in peripheral reticulocytes. Erythroid LOX, which like other lipoxygenases catalyses the degradation of lipids, is unique in its ability to attack intact phospholipids and is the main factor responsible for the degradation of mitochondria during reticulocyte maturation. Strikingly, rabbit erythroid LOX mRNA has 10 tandem repeats of a slightly varied, pyrimidine‐rich 19 nt motif in its 3′‐untranslated region (3′‐UTR). In this study we demonstrate, using gel retardation and UV‐crosslinking assays, that this 3′‐UTR segment specifically binds a 48 kDa reticulocyte protein. Furthermore, the interaction between the 3′‐UTR LOX repeat motif and the 48 kDa protein, purified to homogeneity by specific RNA chromatography, is shown to be necessary and sufficient for specific translational repression of LOX as well as reporter mRNAs in vitro. To our knowledge this is the first case in which translation, presumably at the initiation step, is regulated by a defined protein‐RNA interaction in the 3′‐UTR.

). Whereas these five residues were quantitatively modified, Arg 303 was asymmetrically dimethylated in <33% of hnRNP K and Arg 287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNAbinding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.Arginine dimethylation is a common post-translational modification in eukaryotes (1-5). The enzymes responsible for this modification are the protein-arginine methyltransferases (PRMTs).3 They are classified in two groups (2). Type I enzymes promote the formation of asymmetricThe known mammalian type I enzymes are PRMT1, PRMT2, PRMT3, PRMT4/coactivator-associated arginine methyltransferase 1, PRMT6, and the recently discovered brain-specific PRMT8 (6 -11). Type II enzymes catalyze the symmetric N G ,NЈ G -dimethylation of arginine residues. PRMT5 and PRMT7 are the mammalian type II enzymes described so far (12)(13)(14). PRMT1, which is predominantly localized to the cytoplasm (15), is thought to account for the generation of ϳ85% of asymmetric dimethylarginine residues (16) Many proteins involved in RNA metabolism like hnRNP A1 (36), hnRNP A2 (37), Sam68 (38), and SAF-A (hnRNP U) (39) contain regions with clustered arginine residues in Arg-Gly-Gly motifs (RGG box) or RG repeats. These arginine residues are typically asymmetrically dimethylated. In addition, asymmetric arginine dimethylation has also been found in clustered RXR motifs (40) and other sequences. Therefore, the prediction of a methylated arginine is difficult. Furthermore, the enzyme responsible for the dimethylation of a particular protein is unknown in many cases, and the substrate specificities of the different methyltransferases remain poorly characterized. The exact knowledge of the methylated arginine residues and their quantitative distribution as well as the identification of the relevant methyltransferases is a prerequisite for the functional analysis of arginine methylation.HnRNP K belongs to the family of heterogeneous nuclear RNPs that participate in the processing of pre-mRNAs and in the export of mRNAs from the nucleus. An N-terminal bipartite nuclear-localization signal and an hnRNP K-specific nuclear shuttling signal confer the capacity for bi-directional transport across the nuclear envelop (41, 42). The cytoplasmic accumulation of hnRNP K is mediated by its Erk-dependent serine phosphorylation (43). In the cytoplasm hnRNP K functions in the post-tr...

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The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 59untranslated region (59UTR) and structured sequence elements within the 39UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 59cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 39UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 59UTR and 39UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 59cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 59UTR and HCV 39UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 59UTR and/or HCV 39UTR, recruits eIF3 and enhances HCV IRES-mediated translation.