Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus
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Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus. / Zhang, Yan; Lin, Jinzhong; Tian, Xuhui; Wang, Yuan; Zhao, Ruiliang; Wu, Chenwei; Wang, Xiaoning; Zhao, Pengpeng; Bi, Xiaonan; Yu, Zhenxiao; Han, Wenyuan; Peng, Nan; Liang, Yun Xiang; She, Qunxin.
I: International Journal of Molecular Sciences , Bind 23, Nr. 15, 8515, 2022.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus
AU - Zhang, Yan
AU - Lin, Jinzhong
AU - Tian, Xuhui
AU - Wang, Yuan
AU - Zhao, Ruiliang
AU - Wu, Chenwei
AU - Wang, Xiaoning
AU - Zhao, Pengpeng
AU - Bi, Xiaonan
AU - Yu, Zhenxiao
AU - Han, Wenyuan
AU - Peng, Nan
AU - Liang, Yun Xiang
AU - She, Qunxin
PY - 2022
Y1 - 2022
N2 - Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4 alpha RNase-dead (dCmr4 alpha) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4 alpha in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4 alpha inhibition. Expression of dCmr4 alpha also strongly inhibited plasmid transformation with Cmr2 alpha(HD) and Cmr2 alpha(Palm) mutants, but the inhibition was diminished in Cmr2 alpha(HD,Palm). Since dCmr4 alpha-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-alpha system in the S. islandicus cells expressing dCmr4 alpha. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.
AB - Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4 alpha RNase-dead (dCmr4 alpha) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4 alpha in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4 alpha inhibition. Expression of dCmr4 alpha also strongly inhibited plasmid transformation with Cmr2 alpha(HD) and Cmr2 alpha(Palm) mutants, but the inhibition was diminished in Cmr2 alpha(HD,Palm). Since dCmr4 alpha-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-alpha system in the S. islandicus cells expressing dCmr4 alpha. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.
KW - CRISPR-Cas system
KW - target RNA cleavage
KW - Cmr4
KW - spatiotemporal regulation of Cmr systems
KW - autoimmunity
KW - RNA-activated DNase
KW - cOA synthesis
KW - Sulfolobales
KW - ADAPTIVE IMMUNE-SYSTEMS
KW - CO-TRANSCRIPTIONAL DNA
KW - CMR COMPLEX
KW - EVOLUTIONARY CLASSIFICATION
KW - SULFOLOBUS
KW - PROTEIN
KW - MECHANISM
KW - DEGRADATION
KW - INTERFERENCE
KW - RECOGNITION
U2 - 10.3390/ijms23158515
DO - 10.3390/ijms23158515
M3 - Journal article
C2 - 35955649
VL - 23
JO - International Journal of Molecular Sciences (Online)
JF - International Journal of Molecular Sciences (Online)
SN - 1661-6596
IS - 15
M1 - 8515
ER -
ID: 317447852