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Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis
O P de Melo Neto1, J A Walker, C M Martins de Sa
1Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Abstract:
The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is 'masked' shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca(2+)-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.
Insights
Poly(A)-binding protein (PABP) levels remain constant during early clam development, but its RNA-binding ability is masked after fertilization. This masking is likely due to mRNA association, impacting translation and RNA stability.
Area of Science:
- Molecular Biology
- Developmental Biology
- RNA Biology
Background:
- The poly(A) tail of eukaryotic messenger RNAs (mRNAs) is crucial for regulating translation and RNA stability via interactions with poly(A)-binding proteins (PABPs).
- The precise function of PABP in the selective polyadenylation/deadenylation and translational control of maternal mRNAs during early embryonic development remains incompletely understood.
Purpose of the Study:
- To investigate the characteristics and RNA-binding properties of PABP during the early developmental stages of the clam Spisula solidissima.
- To elucidate the mechanisms underlying PABP regulation, specifically the 'masking' of its RNA-binding potential after fertilization.
Main Methods:
- UV-crosslinking assays to detect PABP-RNA interactions.
- Immunoblotting assays to characterize PABP levels and presence.
- Treatment of cell lysates with guanidinium hydrochloride or micrococcal nuclease to assess PABP RNA-binding potential.
Main Results:
- A single, 70 kDa PABP, highly homologous to known PABPs, was identified in Spisula solidissima oocytes.
- PABP levels were constant throughout early embryogenesis, but its ability to crosslink to poly(A) was masked post-fertilization until the larval stage.
- PABP's full RNA-binding capacity could be restored in embryo lysates via denaturation or controlled nuclease treatment.
Conclusions:
- The masking of PABP's RNA-binding ability in early clam development is likely caused by its association with newly synthesized mRNAs, preventing binding to poly(A) probes.
- This masking phenomenon coincides with cytoplasmic polyadenylation and may play a role in regulating maternal mRNA fate.
- The developed unmasking method holds potential for studying other RNA-binding proteins involved in developmental processes.