Polyribosome analysis for investigating mRNA translation in Xenopus oocytes, eggs and embryos.
The earliest stages of animal development occur without the benefit of zygotic transcription. The absence of transcription necessitates that all changes in the levels of specific proteins must be controlled by post-transcriptional mechanisms, such as the regulated translation of stored maternal mRNAs. One of the major challenges to investigating translational mechanisms is the availability of reliable methods for assaying the translational state of specific mRNAs. The most definitive assay of an mRNA's translational state is polyribosome association; mRNAs actively translated are engaged with polyribosomes while mRNAs translationally repressed are not. While linear gradient centrifugation is commonly used to purify polyribosomes from a wide variety of cell types in different organisms, the isolation of polyribosomes from Xenopus oocytes, eggs and embryos presents some unique challenges. Here we detail the methodology for the isolation and analysis of polyribosomes from Xenopus oocytes, eggs and embryos using step gradient centrifugation. We present detailed protocols, describe the critical controls and provide several examples to guide the interpretation of experimental results regarding the translational state of specific mRNAs.
Sheets
M D
MD
University of Wisconsin School of Medicine and Public Health, Department of Biomolecular Chemistry, Madison, WI 53706, USA. mdsheets@wisc.edu
Fritz
B
B
Hartley
R S
RS
Zhang
Y
Y
D000818:Animals; D015509:Developmental Biology / Q000379:methods*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014981:Xenopus
D012333:RNA, Messenger; D012313:RNA