Poly(A)-associated RNA from the mitochondrial fraction of the fungus Trichoderma

Abstract

Total RNA was extracted from purified mitochondrial and cytoplasmic fractions of germinating conidia of Trichoderma viride and bound to oligo(dT)-cellulose at 22 and 4 degrees C. Under chromatographic conditions which retained very short poly(A) segments (i.e., 4 degrees C), up to 10% of short-term 32PO4-lebeled RNA from the mitochondrial fraction were selectively bound. The poly(A)-associated RNAs from the mitochondrial and cytoplasmic fractions showed the following characteristics. (a) On polyacrylamide gels mitochondrial fraction RNA had a distinctive pattern with a major peak at about 22 S and a smaller one at about 29 S; in contrast, cytoplasmic fraction RNA was heterogenously distributed along the gel. (b) The poly(A) segment released by RNAase digestion of mitochondrial fraction poly(A)-associated RNA migrated on polyacrylamide gels as molecules 20-25-nucleotides long, while that of the cytoplasmic fraction showed an apparent size of 50-60 nucleotides. (c) Mitochondrial fraction RNA bound to oligo(dT)-cellulose in the cold had a guanine + cytosine content of 21% versus 34% for bulk mitochondrial RNA and 48% for cytoplasmic poly(A)-associated RNA; the oligo(dT)-bound RNAs were further identified by their high percentages of adenine residues (46% for the mitochondria and 30% for the cytoplasm). (d) The poly(A)-associated RNA fraction was translated, in vitro, in a cell-free protein-synthesizing system from wheat germ. The products induced by cytoplasmic RNA showed a complex pattern on polyacrylamide gels of many polypeptides ranging in molecular weights from 10000 to 40000. The pattern induced by mitochondrial fraction RNA however, was much simpler, revealing two discrete, main products: a major one at Mr approximately 13000 and a minor one at Mr approximately 20000.