Caspase-3 cleaves hnRNP K in erythroid differentiation.

Post-transcriptional control of gene expression is crucial for the control of cellular differentiation. Erythroid precursor cells loose their organelles in a timely controlled manner during terminal maturation to functional erythrocytes. Extrusion of the nucleus precedes the release of young reticulocytes into the blood stream. The degradation of mitochondria is initiated by reticulocyte 15-lipoxygenase (r15-LOX) in mature reticulocytes. At that terminal stage the release of r15-LOX mRNA from its translational silenced state induces the synthesis of r15-LOX. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator of r15-LOX mRNA translation. HnRNP K that binds to the differentiation control element (DICE) in the 3' untranslated region (UTR) inhibits r15-LOX mRNA translation initiation. During erythroid cell maturation, activation of r15-LOX mRNA translation is mediated by post-translational modifications of hnRNP K and a decrease of the hnRNP K level. To further elucidate its function in the post-transcriptional control of gene expression, we investigated hnRNP K degradation employing an inducible erythroid cell system that recapitulates both nuclear extrusion and the timely controlled degradation of mitochondria, mediated by the activation of r15-LOX synthesis. Interestingly, we detected a specific N-terminal cleavage intermediate of hnRNP K lacking DICE-binding activity that appeared during erythroid differentiation and puromycin-induced apoptosis. Employing mass spectrometry and enzymatic analyses, we identified Caspase-3 as the enzyme that cleaves hnRNP K specifically. In vitro studies revealed that cleavage by Caspase-3 at amino acids (aa) D334-G335 removes the C-terminal hnRNP K homology (KH) domain 3 that confers binding of hnRNP K to the DICE. Our data suggest that the processing of hnRNP K by Caspase-3 provides a save-lock mechanism for its timely release from the r15-LOX mRNA silencing complex and activation of r15-LOX mRNA synthesis in erythroid cell differentiation.

Evidence that fragile X mental retardation protein is a negative regulator of translation.

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.

Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining.

15-lipoxygenase (LOX) expression is translationally silenced in early erythroid precursor cells by a specific mRNA-protein complex formed between the differentiation control element in the 3' untranslated region (UTR) and hnRNPs K and E1. The 3'UTR regulatory complex prevents translation initiation by an unknown mechanism. We demonstrate that the 40S ribosomal subunit can be recruited and scan to the translation initiation codon even when the silencing complex is bound to the 3'UTR. However, the joining of the 60S ribosomal subunit at the AUG codon to form a translation competent 80S ribosome is inhibited, unless initiation is mediated by the IGR-IRES of the cricket paralysis virus. These findings identify the critical step at which LOX mRNA translation is controlled and reveal that 60S subunit joining can be specifically regulated.

mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end.

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.

Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region.

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.