XB-ART-51363J Vis Exp September 27, 2015; (103):
Xenopus laevis as a Model to Identify Translation Impairment.
Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.
PubMed ID: 26437037
PMC ID: PMC4692622
Article link: J Vis Exp
Species referenced: Xenopus
Genes referenced: ccna1 eif2ak3 eif2b1 eif4g1 mapk1 mos
Antibodies: Aurka Ab2 Aurka Ab5 Mapk1 Ab12 Mapk1 Ab13 Mos Ab2 Rps6ka1 Ab1 V5 Ab2
Disease Ontology terms: neurodegenerative disease
References [+] :
Au, Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses. 2010, Pubmed, Xenbase