Related Experiment Videos
Multiple modifications in the phosphoproteins bound to stored messenger RNA in Xenopus oocytes
A Cummings1, P Barrett, J Sommerville
1Department of Biology, University of St. Andrews, U.K.
Abstract:
Messenger RNA molecules accumulated in amphibian oocytes are stabilized and blocked from translation through association with a defined set of phosphoproteins. Phosphoproteins of 60 kDa and 56 kDa (pp60 and pp56) isolated from messenger ribonucleoprotein particles of Xenopus laevis oocytes can be bound in vitro to mRNA sequences. After phospholabelling in vitro, both pp60 and pp56 show a range of ionic forms, which resolve on two-dimensional gel electrophoresis as a series of pairs with identical charge. The similarities between pp60 and pp56 in their ionic properties suggest a common protein primary structure. This suggestion gains further support from proteinase digestion analysis of pp60 and pp56: practically identical size patterns of phospholabelled fragments are generated using a range of different proteinases. However, in spite of their structural similarities, pp60 and pp56 are recognised as antigenically distinct from each other by using polyclonal antibodies. It is concluded from these, and other, observations that pp60 and pp56 are members of a family of structurally similar polypeptides which are subjected to multiple secondary modifications. Of these modifications, phosphorylation appears to be instrumental in establishing tight binding to mRNA, while antigenicity appears to be determined by some other modification. The role of microheterogeneity in the structure of RNA-binding proteins is discussed in relation to the differential activation of mRNA sequences for translation during early development.
Insights
Two Xenopus laevis oocyte phosphoproteins, pp60 and pp56, bind mRNA. Despite structural similarities, phosphorylation aids mRNA binding, while other modifications determine distinct antigenicity, suggesting a family of modified RNA-binding proteins.
Area of Science:
- Molecular Biology
- Developmental Biology
- Biochemistry
Background:
- Messenger RNA (mRNA) in amphibian oocytes is stored in a translationally repressed state.
- This storage involves association with specific phosphoproteins within messenger ribonucleoprotein particles (mRNPs).
Purpose of the Study:
- To investigate the structural and functional relationship between two specific phosphoproteins, pp60 and pp56, involved in mRNA storage in Xenopus laevis oocytes.
- To elucidate the roles of phosphorylation and other modifications in mRNA binding and antigenicity of these proteins.
Main Methods:
- Isolation and in vitro binding of pp60 and pp56 to mRNA sequences.
- Phosphorylation labeling and analysis using two-dimensional gel electrophoresis.
- Proteinase digestion analysis of phospholabeled proteins.
- Immunological characterization using polyclonal antibodies.
Main Results:
- pp60 and pp56 bind to mRNA sequences in vitro.
- Both proteins exhibit similar ionic properties and generate nearly identical phospholabeled fragment patterns after proteinase digestion, suggesting a common primary structure.
- Despite structural similarities, pp60 and pp56 are antigenically distinct.
- Phosphorylation appears crucial for tight mRNA binding, while antigenicity is influenced by other modifications.
Conclusions:
- pp60 and pp56 are part of a protein family with similar structures but distinct antigenic properties due to post-translational modifications.
- Phosphorylation is key for mRNA binding, while other modifications likely dictate antigenic differences.
- Microheterogeneity in RNA-binding proteins may regulate differential mRNA translation during early development.