Measuring CPEB-mediated cytoplasmic polyadenylation-deadenylation in Xenopus laevis oocytes and egg extracts.
The regulation of poly(A) tail length is one important mechanism for controlling gene expression during early animal development. In Xenopus oocytes, the polyadenylation-deadenylation of several essential dormant mRNAs, including cyclin B1 mRNA, are controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA through their associations with protein factors CPEB and CPSF, respectively. CPE-containing, as well as CPE-lacking, pre-mRNAs acquire long poly(A) tails in the nucleus; after their export to the cytoplasm, there is subsequent deadenylation of CPE-containing mRNAs that is controlled by the CPEB-associated factor PARN, a poly(A)-specific ribonuclease. In general, re-adenylation after meiotic maturation of CPE-containing mRNAs is mediated by Gld2, a poly(A) polymerase. Moreover, embryonic poly(A)-binding protein, ePAB, is required for the subsequent elongation and stabilization of the poly(A) tail against PARN and other deadenylating enzymes. In this chapter, we present detailed information for measuring CPEB-mediated cytoplasmic polyadenylation-deadenylation in Xenopus laevis oocytes and egg extracts.
Kim
Jong Heon
JH
Research Institute, National Cancer Center, Goyang, Gyeonggi, South Korea.
Richter
Joel D
JD
D000225:Adenine / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D019926:Cyclin B / Q000032:analysis / Q000378:metabolism; D056744:Cyclin B1; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D009713:Nucleotidyltransferases / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D026723:Polyadenylation; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000378:metabolism*
D002457:Cell Extracts; C490388:Cpeb1 protein, Xenopus; D019926:Cyclin B; D056744:Cyclin B1; D014157:Transcription Factors; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011061:Poly A; D009713:Nucleotidyltransferases; D000225:Adenine