Abstract
During C. elegans apoptosis, the dicer ribonuclease (DCR-1) is cleaved by the cell death protease CED-3 to generate a truncated DCR-1 (tDCR-1) with one and a half ribonuclease III (RNase III) domains, converting it into a deoxyribonuclease (DNase) that initiates apoptotic chromosome fragmentation. We performed biochemical and functional analyses to understand this unexpected RNase to DNase conversion. In full-length DCR-1, tDCR-1 DNase activity is suppressed by its N-terminal DCR-1 sequence. However, not all the sequence elements in the N-terminal DCR-1 are required for this suppression. Our deletion analysis reveals that a 20-residue α-helix sequence in DCR-1 appears to define a critical break point for the sequence required for suppressing tDCR-1 DNase activity through a structure-dependent mechanism. Removal of the N-terminal DCR-1 sequence from tDCR-1 activates a DNA-binding activity that also requires the one half RNase IIIa domain, and enables tDCR-1 to process DNA. Consistently, structural modeling of DCR-1 and tDCR-1 suggests that cleavage of DCR-1 by CED-3 may cause a conformational change that allows tDCR-1 to bind and process DNA, and may remove steric hindrance that blocks DNA access to tDCR-1. Moreover, a new DNase can be engineered using different RNase III domains, including the one from bacterial RNase III. Our results indicate that very distantly related RNase III enzymes have the potential to cleave DNA when processed proteolytically or paired with an appropriate partner that facilitates binding to DNA. We suggest the possibility that this phenomenon may be extrapolated to other ribonucleases.
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Acknowledgements
We thank T Blumenthal, N Pace, and M Yarus (University of Colorado) for comments and members of the Xue lab for helpful discussions. Computational resources used in this study were supported by Tsinghua National Laboratory for Information Science and Technology. This work was supported by a Tsinghua-Peking University Life Science Center fund, the 973 Program 2013CB945602, and NIH grants R01 GM59083 and R01 GM79097.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
CED-3 protease cleavage assays for different DCR-1 mutants. (PDF 261 kb)
Supplementary information, Figure S2
In vitro DNase activity assays of DCR-1 proteins. (PDF 138 kb)
Supplementary information, Figure S3
DNase activity assays of DCR-1(1-1845; Δ1385-1404) and DCR-1(1-1384-GS-1405-1845). (PDF 107 kb)
Supplementary information, Figure S4
DNA binding assays. (PDF 103 kb)
Supplementary information, Figure S5
RNase activity assays of various DCR-1 truncations. (PDF 86 kb)
Supplementary information, Figure S6
Comparison of modeled DCR-1 structures with the structure of Giardia Dicer. (PDF 111 kb)
Supplementary information, Table S1
Rescue of the vulva defects of dcr-1(ok247) animals by expression of various DCR-1 truncations. (PDF 64 kb)
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Ge, X., Zhao, X., Nakagawa, A. et al. A novel mechanism underlies caspase-dependent conversion of the dicer ribonuclease into a deoxyribonuclease during apoptosis. Cell Res 24, 218–232 (2014). https://doi.org/10.1038/cr.2013.160
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DOI: https://doi.org/10.1038/cr.2013.160
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