Exploring Translational Control of Maternal mRNAs in Zebrafish

Methods Mol Biol. 2021;2218:367-380. doi: 10.1007/978-1-0716-0970-5_29.

Abstract

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

Keywords: Cytoplasmic polyadenylation; Maternal RNA; Polysome profiling; Translational regulation; Zebrafish.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytoplasm / genetics
  • Embryo, Nonmammalian / physiology
  • Embryonic Development / genetics
  • Oocytes / physiology
  • Oogenesis / genetics
  • Poly A / genetics
  • Polyadenylation / genetics
  • Protein Biosynthesis / genetics*
  • RNA, Messenger, Stored
  • Zebrafish / genetics*

Substances

  • RNA, Messenger, Stored
  • Poly A