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.

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.

Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.