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Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes.

ABSTRACT: Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.

SUBMITTER: Ota R

PROVIDER: S-EPMC3024781 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes.

Ota Ryoma R Kotani Tomoya T Yamashita Masakane M

The Journal of biological chemistry 20101123 4

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the trans ...[more]

PMID: 21098481

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Project description:Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that synaptosomes purified from rodent and human frozen brain (control and Alzheimer disease) can be studied both biochemically and functionally. This method opens the way to directly testing the effects of novel drugs on native ion channels.

Dataset Information

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes.

Publications

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes.

Similar Datasets

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