Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: Proteins associated with the cytoplasmic polysomal and non-polysomal mRNAs (messenger ribonucleoprotein complex)

Cytoplasmic polysomal and non-polysomal mRNA-associated protein complexes were isolated from, and characterized in, developing somatic and zygotic embryos of alfalfa (Medicago sativa L.). S-35-methionine-labelled intact embryos were irradiated with ultraviolet light (UV) in situ to cross-link mRNA and proteins occurring within one bond length, and the polysomal and non-polysomal fractions were extracted. Then the mRNA-protein complexes were isolated from the fractions and separated using two cycles of affinity chromatography on an oligo(dT)-cellulose column. Following digestion with RNase A and T1 and micrococcal nuclease, mRNA-associated proteins were separated by sos-PAGE. Several polypeptides of 15-150 kDa were associated with the polysomal and non-polysomal (ribonucleoprotein, mRNP) fractions of alfalfa embryos after UV-cross-linking. Many of the polypeptides associated with the polysomal and non-polysomal mRNAs were qualitatively similar, although their concentration in the two fractions was different. However, some developmentally stage-specific polypeptides were found to be associated with the non-polysomal mRNA fraction during the early stages of embryogenesis (pre-cotyledonary) of somatic embryos. Thus the presence of mRNPs during embryogenesis has been demonstrated, and proteins intimately associated with the mRNAs identified.