Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis.
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.
de Melo Neto
O P
OP
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Walker
J A
JA
Martins de Sa
C M
CM
Standart
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D049872:Bivalvia / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002457:Cell Extracts; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D005306:Fertilization; D018507:Gene Expression Regulation, Developmental; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010063:Ovum / Q000166:cytology / Q000378:metabolism; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D014466:Ultraviolet Rays
D002457:Cell Extracts; D039102:Poly(A)-Binding Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011061:Poly A; D008836:Micrococcal Nuclease; D019791:Guanidine