DEGRADATION OF THE SOYBEAN RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE SMALL-SUBUNIT MESSENGER-RNA, SRS4, INITIATES WITH ENDONUCLEOLYTIC CLEAVAGE

The degradation of the soybean SRS4 mRNA, which encodes the small subu nit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro , These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced mo re rapidly than others. Comparison of sizes of products on Northern (R NA) blots showed that the combined sizes of pairs of proximal and dist al products form contiguous full-length SRS4 mRNAs, When the 3' ends o f the proximal products and the 5' ends of the distal products were ma pped by S1 nuclease and primer extension assays, respectively, both se ts of ends mapped to the same sequences within the SRS4 mRNA, A small in vitro-synthesized RNA fragment containing one cleavage site inhibit ed cleavage of all major sites, equivalently consistent with one enzym atic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequenc e was found among several cleavage sites, Preliminary evidence suggest ed that secondary structure could play a role in site selection, The s tructures of the 5' ends of the proximal products and the 3' ends of t he distal products were examined. Proximal products were found with ap proximately equal frequency in both m(7)G cap(+) and m(7)G cap(-) frac tions, suggesting that the endonucleolytic cleavage events occurred in dependently of the removal of the 5' cap structure, Distal products we re distributed among fractions with poly(A) tails ranging from undetec table to greater than 100 nucleotides in length, suggesting that the e ndonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease mod el in which an endonucleolytic cleavage event is the initial step in S RS4 mRNA degradation.