Poly(A) metabolism in the messenger RNA of Physarum polycephalum



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Correlations between mRNA longevity and poly(A) metabolism have been investigated in the acellular slime mold Physarum polycephalum. The kinetics of appearance of poly(A)-containing RNA in the nucleus and cytoplasm have been investigated and discussed in relation to current theories on the transcription, polyadenylation, processing, and transport of heterogeneous nuclear RNA and messenger RNA. The kinetics of labeling were found to support the current theory of the precursor-product relationship between hnRNA and cytoplasmic messenger RNA. Nuclear and cytoplasmic poly(A) were found to contain two major poly(A) segments. The larger segment was estimated to have an average size of about 200 nucleotides (N) in the nucleus and about 125(N) immediately after its appearance in the cytoplasm, indicating cleavage of poly(A) may occur upon transport to the cytoplasm. The smaller segment had an average size of 18N both in the nucleus and cytoplasm and is thus referred to as oligo(A). Experiments involving different mechanisms of cleavage of poly(A) from mRNA were conducted to probe the relationship between the oligo(A) and poly(A) sequences in Physarum mRNA. The results indicated that, unlike the poly(A) sequences in the mRNA of Dictyostelium discoideum, if the segments exist on the same mRNA molecule, they are separated by a nucleotide tract containing at least one guanylic acid residue. Analysis of poly(A) in the nucleus and cytoplasm during sclerotium formation revealed the existence of very large classes of poly(A) roughly twice as long as those present under normal conditions. The sequences are probably synthesized de novo in the nucleus and transported to the cytoplasm. Analysis of cytoplasmic degradation of poly(A) has also been investigated. Under normal conditions, poly(A) degradation occurs with biphasic kinetics. Using electrophoresis data the first phase was thought to correlate with the degradation of the larger poly(A) segment, and the second more stable phase, degradation of oligo(A). Average half lives of 5 and 29 hours respectively were calculated for each sequence. Degradation of the larger segment is thought to occur by two processes; a shortening process which is responsible for the size decrease, and a destruction process which rapidly and completely destroys the segment. Both processes appear to be underway immediately after the beginning of the chase and may begin when the mRNA first enters the cytoplasm. The steady state profile of the larger segment was found to be relatively homogeneous. This, along with electrophoresis data on labeled poly(A), indicate that the shortening process terminates before the destructive process. Further evidence for the existence of two poly(A) degradative processes was obtained in an analysis of poly(A) degradation in the presence of various protein synthesis inhibitors. When inhibitors were used which interfere with the translocation step of protein synthesis but leave the mRNA associated with the polyribosome (cycloheximide or emetine), the poly(A) shortening process appeared to continue normally whereas the poly(A) destruction process was inhibited. Protein synthesis inhibitors (heat shock, puromycin) resulting in dissociation of the polysome had no effect on either process. Normal degradation thus may require release of the mRNA from the polyribosome. The lack of poly(A) destruction in the presence of cycloheximide and emetine indicates the resistance to degradation is not caused by depression of the synthesis of a labile poly(A) degrading enzyme. Sucrose gradient analysis of Physarum lysates indicated that some of the poly(A) exists in association with proteins in a complex that sedimented about 12S and could be disrupted with SDS. In pulse-chase experiments the non-complexed poly(A) decayed with a half-life equal to that of the larger poly(A) segment whereas the complexed material remained stable during the 6 hours of the experiment. The complexed material may thus be oligo(A) and the free poly(A), the larger segment. Further support for this was found when the degradation of the free poly(A) was suppressed in the presence of cycloheximide. The kinetics of poly(A)-RNA turnover has also been investigated using pulse-chase techniques. In both starved and normal plasmodia the kinetics of poly(A)-RNA degradation is biphasic. A significant stabilization of at least one class of poly(A)-RNA is seen to occur in Physarum induced to undergo sclerotium formation.