CUSATIVIN, A NEW CYTIDINE-SPECIFIC RIBONUCLEASE ACCUMULATED IN SEEDS OF CUCUMIS-SATIVUS L

Dry seeds of Cucumis sativus L. were found to contain a heat-sensitive endoribonuclease of a novel type which we have named cusativin. It wa s purified to apparent electrophoretic homogeneity by chromatography t hrough S-Sepharose Fast Flow, Sephadex G-75, CM-Sepharose, Superdex 75 -FPLC (fast protein liquid chromatography) and Mono S-FPLC. It is a si ngle unglycosylated polypeptide chain with an apparent molecular mass (M(r)) of 22900. Polyclonal anti-cusativin antibodies raised in rabbit s only reacted with melonin, the translation inhibitor from Cucumis me lo L. Functional, Western blot and enzyme-linked immunosorbent assay ( ELISA) analyses indicated that cusativin is present in the coat and co tyledons of dry seeds, but not in embryonic axes. Cusativin is accumul ated in maturing seeds. By contrast, after seed germination there is d egradation of the cusativin present in cotyledons but not that present in the seed coat. The preference of cusativin for polynucleotide clea vage was poly(C)much greater than poly(A) acids, poly(U) and poly(G) b eing unaffected by cusativin. Under the denaturing conditions used for RNA sequencing, cusativin acted only on poly(C). Cusativin proved to be useful for RNA sequencing, in particular, complementing the data ob tained with RNase CL3. Cusativin represents a new class of plant RNase and, as far as we are aware, is the first plant enzyme that shows cle avage specificity for cytidine under the denaturing conditions of RNA sequencing.