Identification and characterization of developmentally regulated mRNP proteins of Dictyostelium discoideum.
The isolation of poly(A)+ polysomal and nonpolysomal RNPs by oligo(dT)-cellulose chromatography has led to the identification of more than 20 polypeptides that bind to the poly(A)+ mRNA in growing Dictyostelium cells. Most of these polypeptides were identified in experiments using short-wave UV light (254 nm) to crosslink specifically bound proteins to the RNA. Digestion of the RNPs with ribonucleases A and T1 prior to their application to oligo(dT)-cellulose permitted the isolation of the 3' poly(A)-protein complexes. In polysomal RNPs, two major polypeptides, with molecular weights of 31,000 (p31) and 31,500 (p31.5), are bound to poly(A). These proteins can also be purified from cytoplasmic extracts by affinity chromatography on poly(A)-Sepharose. Partial proteolytic digestion of p31 and p31.5 indicates that they are closely related. The UV-crosslinking experiments established that p31 and p31.5 bind to the non-poly(A) segments of mRNA as well. In nonpolysomal RNPs, p31 and a polypeptide with a molecular weight of 29,500 (p29.5) are the major species associated with poly(A). Partial proteolytic digestion of p29.5 indicates that it is closely related to p31 and p31.5. Only small amounts of p29.5 were observed in the polysomal poly(A)-protein complexes. Early in Dictyostelium development, when cellular translation activity is sharply reduced, most of the p29.5, p31 and p31.5 present is selectively degraded. These observations are consistent with a translational role for these proteins.
D002846:Chromatography, Affinity; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000528:radiation effects; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012685:Sepharose; D013347:Subcellular Fractions / Q000032:analysis; D014466:Ultraviolet Rays
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C026452:messenger ribonucleoprotein; C027903:oligo (dT); D011061:Poly A; D012313:RNA; D012685:Sepharose