Abstract
Dry seeds of Cucumis sativus L. were found to contain a heat-sensitive endoribonuclease of a novel type which we have named cusativin. It was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75, CM-Sepharose, Superdex 75-FPLC (fast protein liquid chromatography) and Mono S-FPLC. It is a single unglycosylated polypeptide chain with an apparent molecular mass (Mr) of 22900. Polyclonal anti-cusativin antibodies raised in rabbits only reacted with melonin, the translation inhibitor from Cucumis melo L. Functional, Western blot and enzyme-linked immunosorbent assay (ELISA) analyses indicated that cusativin is present in the coat and cotyledons of dry seeds, but not in embryonic axes. Cusativin is accumulated in maturing seeds. By contrast, after seed germination there is degradation of the cusativin present in cotyledons but not that present in the seed coat. The preference of cusativin for polynucleotide cleavage was poly(C)≫poly(A) acids, poly(U) and poly(G) being 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 obtained 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 cleavage specificity for cytidine under the denaturing conditions of RNA sequencing.
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Abbreviations
- ELISA:
-
enzyme-linked immunosorbent assay
- Mra:
-
molecular mass
- RIP:
-
ribosome-inactivating protein
- rRNA:
-
ribosomal ribonucleic acid
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This work was supported by grants from Comisión Interministerial de Ciencia y Technologia (CICYT; BIO89-0705 and BIO92-0231), the Junta de Castilla y León to T. Girbés and from the CICYT (PM89-0102) and Fondo de Investigaciones Sanitarias (FIS) 89-0858 to E. Mendez. M.A. Rojo and F.J. Arias hold predoctoral fellowships from Iberdrola S.A. We wish to thank N. Skinner (University of Salamanca, Spain) for correcting the English version of the manuscript.
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Rojo, M.A., Arias, F.J., Iglesias, R. et al. Cusativin, a new cytidine-specific ribonuclease accumulated in seeds of Cucumis sativus L.. Planta 194, 328–338 (1994). https://doi.org/10.1007/BF00197532
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DOI: https://doi.org/10.1007/BF00197532