Abstract
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.
Highlights
Translation inhibition is abolished by phosphorylation of Y458 in KH domain 3 that mediates binding to the DICE in the r15-LOX mRNA 30UTR.[4,5]
The analysis of proteins involved in r15-LOX mRNA translational control revealed that the level of hnRNP K decreases during erythroid differentiation of K562 cells[9] (Figure 1a)
No peptides corresponding to KH domain 3 that mediates binding to the DICE in the r15-LOX mRNA5 were detected (Figure 1d)
Summary
Regulates translation of specific mRNAs. Post-translational modifications of hnRNP K have been shown to modulate its capacity in regulatory complex formation.[3,4,5,6] Erythroid precursor cells undergo nuclear extrusion and mitochondria degradation in reticulocytes at the terminal step of erythrocyte formation. A decreasing hnRNP K level contributes to the release of the silencing complex.[9] the function of site-specific phosphorylation in r15-LOX mRNA translation regulation has been elucidated, there is no information about the mechanism of hnRNP K degradation in erythroid differentiation. Caspases catalyze site-specific protein cleavage in apoptosis,[11] but were shown to be activated in terminal erythroid differentiation,[12,13,14] which is not associated with apoptosis.[15] Interestingly, a specific N-terminal hnRNP K fragment that migrates at 48 kD accumulates during erythroid. Residues D334–G335 were identified as Caspase-3 cleavage site that separates the DICE-binding KH domain 3 from the N-terminal part, which contains critical protein-protein interaction domains.[5,16,17] Caspase-3 mediated cleavage inactivates hnRNP K as a regulator of r15-LOX mRNA translation
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