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

Author： Pramanik Saroj K. Bewley J. Derek

Publisher： Oxford University Press

ISSN： 1460-2431

Source： Journal of Experimental Botany, Vol.47, Iss.12, 1996-12, pp. : 1871-1879

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Abstract

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.). ³⁵S-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 SDS-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 (precotyledonary) of somatic embryos. Thus the presence of mRNPs during embryogenesis has been demonstrated, and proteins intimately associated with the mRNAs identified.