A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins.
A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins.
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DOI：
10.1038/s41598-023-36969-6
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发表时间：
2023-07-03
影响因子：
4.6
通讯作者：
Hayes, Michael L.
中科院分区：
文献类型：
--
作者：
Boyd, Robert D.;Hayes, Michael L.
植物细胞器中的C-to-U RNA编辑发生在由各种类型的核编码蛋白组成的复合物中。DYW-脱氨酶是催化C至U修饰编辑所需的水解脱氨的锌金属酶。DYW-脱氨酶结构域的解析晶体结构显示与典型胞苷脱氨机制一致的所有结构特征。然而，一些来自植物的重组DYW-脱氨酶已经与体外核糖核酸酶活性相关。编辑因子的直接核糖核酸酶活性是混淆的，因为它不是胞嘧啶脱氨基所必需的，理论上对mRNA编辑是有害的，并且在体内没有明确的生理功能。表达了带有His标签的重组拟南芥DYW 1（rAtDYW 1），并利用固定化金属亲和层析（IMAC）纯化。在不同的条件下，将荧光标记的RNA寡核苷酸与重组AtDYW 1孵育。在多个时间点从一式三份反应记录RNA探针的相对切割百分比。用锌螯合剂EDTA和1，10-菲咯啉处理rAtDYW 1的效果进行了检查。重组His标记的RNA编辑因子AtRIP2、ZmRIP9、AtRIP9、AtOZ1、AtCRR 4和AtORR 1在E. coli中表达并纯化。在不同编辑因子存在下测定rAtDYW 1的核糖核酸酶活性。最后，研究了核苷酸和修饰核苷对核酸酶活性的影响。本研究中观察到的RNA切割与体外重组编辑因子rAtDYW 1有关。裂解反应对高浓度的锌螯合剂敏感，表明锌离子对活性的作用。添加等摩尔浓度的重组RIP/MORF蛋白降低了与rAtDYW 1相关的切割活性。然而，添加等摩尔浓度的纯化的重组编辑复合物蛋白AtCRR 4，AtORRM 1和AtOZ 1没有强烈抑制核糖核酸酶活性的RNA缺乏AtCRR 4顺式元件。虽然AtCRR 4抑制AtDYW 1活性的寡核苷酸同源顺式元件。编辑因子在体外限制rAtDYW 1的核糖核酸酶活性的观察结果表明，在没有天然编辑复合物伴侣的情况下，核酸酶活性仅限于RNA。纯化的rAtDYW 1在体外与RNA水解相关，并且活性被RNA编辑因子特异性抑制。
Organellar C-to-U RNA editing in plants occurs in complexes composed of various classes of nuclear-encoded proteins. DYW-deaminases are zinc metalloenzymes that catalyze hydrolytic deamination required for C-to-U modification editing. Solved crystal structures for DYW-deaminase domains display all structural features consistent with a canonical cytidine deamination mechanism. However, some recombinant DYW-deaminases from plants have been associated with ribonuclease activity in vitro. Direct ribonuclease activity by an editing factor is confounding since it is not required for deamination of cytosine, theoretically would be inimical for mRNA editing, and does not have a clear physiological function in vivo. His-tagged recombinant DYW1 from Arabidopsis thaliana (rAtDYW1) was expressed and purified using immobilized metal affinity chromatography (IMAC). Fluorescently labeled RNA oligonucleotides were incubated with recombinant AtDYW1 under different conditions. Percent relative cleavage of RNA probes was recorded at multiple time points from triplicate reactions. The effects of treatment with zinc chelators EDTA and 1, 10-phenanthroline were examined for rAtDYW1. Recombinant His-tagged RNA editing factors AtRIP2, ZmRIP9, AtRIP9, AtOZ1, AtCRR4, and AtORRM1 were expressed in E. coli and purified. Ribonuclease activity was assayed for rAtDYW1 in the presence of different editing factors. Lastly, the effects on nuclease activity in the presence of nucleotides and modified nucleosides were investigated. RNA cleavage observed in this study was linked to the recombinant editing factor rAtDYW1 in vitro. The cleavage reaction is sensitive to high concentrations of zinc chelators, indicating a role for zinc ions for activity. The addition of equal molar concentrations of recombinant RIP/MORF proteins reduced cleavage activity associated with rAtDYW1. However, addition of equal molar concentrations of purified recombinant editing complex proteins AtCRR4, AtORRM1, and AtOZ1 did not strongly inhibit ribonuclease activity on RNAs lacking an AtCRR4 cis-element. Though AtCRR4 inhibited AtDYW1 activity for oligonucleotides with a cognate cis-element. The observation that editing factors limit ribonuclease activity of rAtDYW1 in vitro, suggests that nuclease activities are limited to RNAs in absence of native editing complex partners. Purified rAtDYW1 was associated with the hydrolysis of RNA in vitro, and activity was specifically inhibited by RNA editing factors.
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DOI：
10.1073/pnas.0307163101
发表时间：
2004-01-06
影响因子：
11.1
作者：
Miyamoto, T;Obokata, J;Sugiura, M
通讯作者：
Sugiura, M
影响因子：
7.2
作者：
Hashimoto, M;Endo, T;Shikanai, T
通讯作者：
Shikanai, T
影响因子：
5.9
作者：
Oldenkott, Bastian;Yang, Yingying;Schallenberg-Ruedinger, Mareike
通讯作者：
Schallenberg-Ruedinger, Mareike
影响因子：
7.2
作者：
Cheng, Shifeng;Gutmann, Bernard;Small, Ian
通讯作者：
Small, Ian
影响因子：
4.5
作者：
Neuwirt, J;Takenaka, M;Brennicke, A
通讯作者：
Brennicke, A