Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 39 end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 39 end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 39 end trimming, which might serve to enhance snoRNA stability.

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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 39 untranslated region (39UTR). 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.

Erythroid precursor cells undergo nuclear extrusion and degradation of mitochondria when they mature to erythrocytes. It has been suggested before that the reticulocyte 15-lipoxygenase (r15-LOX) plays an important role in initiating the breakdown of mitochondria in rabbit reticulocytes. The expression of rabbit r15-LOX is regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) K and E1 at the translational level. However, this mechanism has never been confirmed in human erythropoiesis. Based on K562 cells we have set up an inducible human erythroid cell system. We show that, during induction, K562 cells exhibit changes in morphology and protein expression that are characteristic for terminal erythroid maturation: nuclear exclusion, expression of endogenous human r15-LOX regulated by hnRNP K and hnRNP E1, and loss of mitochondria. Importantly, induction of terminal erythroid maturation in primary human CD34؉ cells recapitulated the results obtained in K562 cells. Employing the physiologically relevant K562 cell system we uncovered a new mechanism of interdependent posttranscriptional regulation of gene expression. The timely expression of the tyrosine kinase c-Src, which phosphorylates hnRNP K in later stages, is controlled by hnRNP K in early stages of erythroid maturation. hnRNP K binds to the 3-untranslated region of the c-Src mRNA and inhibits its translation by blocking 80 S ribosome formation. In premature erythroid cells, small interfering RNA-mediated knockdown of hnRNP K, but not of hnRNP E1, leads to the de-repression of c-Src synthesis.

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