ABSTRACT
The neurexin genes (NRXN1, NRXN2, and NRXN3) encode polymorphic presynaptic proteins that are implicated in synaptic plasticity and memory processing. In rat brain neurons grown in culture, depolarization induces reversible, calcium-dependent, repression of NRXN2α exon 11 (E11) splicing. Using Neuro2a cells as a model, we explored E11 cis elements and trans-acting factors involved in alternative splicing of NRXN2α E11 pre-mRNA under basal and depolarization conditions. E11 mutation studies revealed two motifs, CTGCCTG (enhancer) and GCACCCA (suppressor) regulating NRXN2α E11 alternative splicing. Subsequent E11 RNA affinity pull-down experiments demonstrated heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP L binding to this exon. Under depolarization, the amount of E11-bound hnRNP L (but not of hnRNP K) increased, in parallel to NRXN2α E11 splicing repression. Depletion of hnRNP K or hnRNP L in the Neuro2a cells by specific siRNAs enhanced NRXN2α E11 splicing and ablated the depolarization-induced repression of this exon. In addition, depolarization suppressed whereas hnRNP K depletion enhanced NRXN2α expression. These results indicate a role for hnRNP K in regulation of NRXN2α expression and of hnRNP L in the activity-dependent alternative splicing of neurexins which may potentially govern trans-synaptic signaling required for memory processing.
Subject(s)
Alternative Splicing , Exons , Nerve Tissue Proteins/genetics , Animals , Cell Line, Tumor , Enhancer Elements, Genetic , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Heterogeneous-Nuclear Ribonucleoprotein L/genetics , Heterogeneous-Nuclear Ribonucleoprotein L/metabolism , Membrane Potentials , Mice , Mutation , Nerve Tissue Proteins/metabolism , Nucleotide Motifs , Protein Binding , RNA, Small Interfering , Regulatory Sequences, Ribonucleic AcidABSTRACT
Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.
