Abstract
Class 1 ribonuclease III (RNase III), found in bacteria and yeast, is involved in processing functional RNA molecules such as ribosomal RNAs (rRNAs). However, in Arabidopsis thaliana, the lack of an obvious phenotype or quantitative change in mature rRNAs in class 1 RNase III (AtRTL2) mutants and overexpressing plants suggests that AtRTL2 is not involved in rRNA maturation. We characterized the in vitro activity of AtRTL2 to consider its in vivo function. AtRTL2 cleaved double-stranded RNA (dsRNA) specifically in vitro, yielding products of approximately 25 nt or longer in length, in contrast to 10–20 nt long products in bacteria and yeasts. Although dsRNA-binding activity was not detected, the dsRNA-binding domains in AtRTL2 were essential for its dsRNA-cleaving activity. Accumulation of small RNAs derived from transgene dsRNAs was increased when AtRTL2 was transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. These results raise the possibility that AtRTL2 has functions distinct from those of other class 1 RNase IIIs in vivo.
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Acknowledgments
The authors thank the Kazusa DNA Research Institute and the Arabidopsis Biological Resource Center for kindly providing the Arabidopsis seeds and cDNA clones, Drs. Yasuhiko Matsushita and Masahiro Kasahara, Tokyo University of Agriculture and Technology, Tokyo, Japan, Dr. Yasuo Niwa, University of Shizuoka, Shizuoka, Japan, and Dr. James C. Carrington, Oregon State University, USA, for kindly providing plasmids, and Dr. David C. Baulcombe, University of Cambridge, UK, for kindly providing N. benthamiana seeds. This work was supported by Grants-in-Aid for Scientific Research (No. 21580411 to T.F. and No. 20580045 to H.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Fig. S1
Phenotypes of AtRTL2 mutant and AtRTL2-overexpressing plants. a Photographs of an A. thaliana WT plant, an AtRTL2 mutant plant and an AtRTL2-overexpressing plant that were grown for six weeks. Both mutant and overexpressing plants did not show abnormal phenotypes. b Northern analysis of AtRTL2 mRNA from leaves of each plant (upper panel). About 10 μg of total RNA was separated on a 1.2% agarose gel and stained by EtBr (lower panel). No quantitative changes in mature rRNAs are shown in each lane. WT, wild type; atrtl2, AtRTL2 T-DNA-insertion mutant (SALK 067855); 35S:AtRTL2, AtRTL2-overexpressing plant. (PPT 328 kb)
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Kiyota, E., Okada, R., Kondo, N. et al. An Arabidopsis RNase III-like protein, AtRTL2, cleaves double-stranded RNA in vitro. J Plant Res 124, 405–414 (2011). https://doi.org/10.1007/s10265-010-0382-x
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DOI: https://doi.org/10.1007/s10265-010-0382-x