Interaction of ZPR1 with Translation Elongation Factor-1α in Proliferating Cells

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1α (eEF-1α). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1α and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1α by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1α restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1α contributes to normal cellular proliferation

A short antisense oligonucleotide masking a unique intronic motif prevents skipping of a critical exon in spinal muscular atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Most SMA cases are associated with the low levels of SMN owing to deletion of Survival Motor Neuron 1 (SMN1). SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Hence, correction of aberrant splicing of SMN2 exon 7 holds the potential for cure of SMA. Here we report an 8-mer antisense oligonucleotide (ASO) to have a profound stimulatory response on correction of aberrant splicing of SMN2 exon 7 by binding to a unique GC-rich sequence located within intron 7 of SMN2. We confirm that the splicing-switching ability of this short ASO comes with a high degree of specificity and reduced off-target effect compared to larger ASOs targeting the same sequence. We further demonstrate that a single low nanomolar dose of this 8-mer ASO substantially increases the levels of SMN and a host of factors including Gemin 2, Gemin 8, ZPR1, hnRNP Q and Tra2-β1 known to be down regulated in SMA. Our findings underscore the advantages and unmatched potential of very short ASOs in splicing modulation in vivo

The cytoplasmic zinc finger protein ZPR1 accumulates in the nucleolus of proliferating cells

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells

Deficiency of the zinc finger protein ZPR1 causes defects in transcription and cell cycle progression

The zinc finger protein ZPR1 is present in both the cytoplasm and nucleoplasm. Cell cycle analysis demonstrates that ZPR1 undergoes major changes in subcellular distribution during proliferation. ZPR1 is diffusely localized throughout the cell during the G(1) and G(2)/M phases of the cell cycle. In contrast, ZPR1 redistributes to the nucleus during S phase and ZPR1 exhibits prominent co-localization with the survival motor neurons protein and the histone gene-specific transcription factor NPAT in subnuclear foci, including Cajal bodies that associate with histone gene clusters. ZPR1 deficiency causes disruption of survival motor neurons and NPAT localization within the nucleus, blocks S phase progression, and arrests cells in both the G(1) and G(2) phases of the cell cycle. These changes in subnuclear architecture and cell cycle progression may be caused by transcriptional defects in ZPR1-deficient cells, including decreased histone gene expression

Inhibition of chloroplast protein phosphorylation by cAMP in Lemna paucicostata 6746

Phosphorylation was carried out in chloroplasts, purified on sucrose density gradient, using [γ-32P] ATP. Among several phosphopolypeptides detected, polypeptides in the Mr range of 24-22 k, which are known to constitute the light-harvesting chlorophyll a/b-binding protein complex (LHCP), and 16-18 k were highly phosphorylated. Cyclic AMP (100 μM) decreased the overall phosphorylation of chloroplast polypeptides and its effect was more striking on LHCP. Other nucleotides such as cGMP, 2',3'-cAMP, 2',3'-cGMP and 5'-AMP, at equimolar level, did not show any significant effect on phosphorylation of chloroplast polypeptides. The effect of cAMP was also studied on light-dependent in vitro phosphorylation of chloroplast polypeptides by incubating a crude preparation of intact chloroplasts with [32Pi]. In dark control, two polypeptides of Mr 66 and 64 k were distinctly phosphorylated. On illumination, several other polypeptides in the range of 97-12 k were also phosphorylated, with the 26-24 k LHCP fraction being the major phosphopolypeptides. Cyclic AMP (at 100 μM and 1 mM) specifically caused a decrease in phosphorylation of the 26-24, 21-20 and 12 k polypeptides. The phosphorylation status of the 66-64 k doublet, as attained in dark, remained unaffected in the presence of light and/or cAMP. Sodium fluoride, a protein phosphatase inhibitor, enhanced the phosphorylation of 26-24, 21-20 and 12 k polypeptides but did not affect the inhibitory action of cAMP. It is thus likely that cAMP down-regulates protein kinase(s) responsible for the phosphorylation of these polypeptides. The present study also provides evidence that this protein kinase is sensitive to staurosporine, a protein kinase inhibitor

Molecular mechanisms of neurodegeneration in spinal muscular atrophy

© the authors, publisher and licensee Llibertas Aacademica Llimited. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA

ZPR1 Is Essential for Survival and Is Required for Localization of the Survival Motor Neurons (SMN) Protein to Cajal Bodies

Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1(−/−) embryos and the amount of cytoplasmic snRNP detected in Zpr1(−/−) embryos was reduced compared with that in wild-type embryos. We found that Zpr1(−/−) mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival

ZPR1 prevents R-loop accumulation, upregulates SMN2 expression and rescues spinal muscular atrophy

© The Author(s) (2019). Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene. A second copy, SMN2, is similar to SMN1 but produces ∼10% SMN protein because of a single-point mutation that causes splicing defects. Chronic low levels of SMN cause accumulation of co-transcriptional R-loops and DNA damage leading to genomic instability and neurodegeneration in SMA. Severity of SMA disease correlates inversely with SMN levels. SMN2 is a promising target to produce higher levels of SMN by enhancing its expression. Mechanisms that regulate expression of SMN genes are largely unknown. We report that zinc finger protein ZPR1 binds to RNA polymerase II, interacts in vivo with SMN locus and upregulates SMN2 expression in SMA mice and patient cells. Modulation of ZPR1 levels directly correlates and influences SMN2 expression levels in SMA patient cells. ZPR1 overexpression in vivo results in a systemic increase of SMN levels and rescues severe to moderate disease in SMA mice. ZPR1-dependent rescue improves growth and motor function and increases the lifespan of male and female SMA mice. ZPR1 reduces neurodegeneration in SMA mice and prevents degeneration of cultured primary spinal cord neurons derived from SMA mice. Further, we show that the low levels of ZPR1 associated with SMA pathogenesis cause accumulation of co-transcriptional RNADNA hybrids (R-loops) and DNA damage leading to genomic instability in SMA mice and patient cells. Complementation with ZPR1 elevates senataxin levels, reduces R-loop accumulation and rescues DNA damage in SMA mice, motor neurons and patient cells. In conclusion, ZPR1 is critical for preventing accumulation of co-transcriptional R-loops and DNA damage to avert genomic instability and neurodegeneration in SMA. ZPR1 enhances SMN2 expression and leads to SMN-dependent rescue of SMA. ZPR1 represents a protective modifier and a therapeutic target for developing a new method for the treatment of SMA

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt

Structural insights into the interaction of the evolutionarily conserved ZPR1 domain tandem with eukaryotic EF1A, receptors, and SMN complexes

Eukaryotic genomes encode a zinc finger protein (ZPR1) with tandem ZPR1 domains. In response to growth stimuli, ZPR1 assembles into complexes with eukaryotic translation elongation factor 1A (eEF1A) and the survival motor neurons protein. To gain insight into the structural mechanisms underlying the essential function of ZPR1 in diverse organisms, we determined the crystal structure of a ZPR1 domain tandem and characterized the interaction with eEF1A. The ZPR1 domain consists of an elongation initiation factor 2-like zinc finger and a double-stranded β helix with a helical hairpin insertion. ZPR1 binds preferentially to GDP-bound eEF1A but does not directly influence the kinetics of nucleotide exchange or GTP hydrolysis. However, ZPR1 efficiently displaces the exchange factor eEF1Bα from preformed nucleotide-free complexes, suggesting that it may function as a negative regulator of eEF1A activation. Structure-based mutational and complementation analyses reveal a conserved binding epitope for eEF1A that is required for normal cell growth, proliferation, and cell cycle progression. Structural differences between the ZPR1 domains contribute to the observed functional divergence and provide evidence for distinct modalities of interaction with eEF1A and survival motor neuron complexes