拟南芥核质转运蛋白及RNA拓扑异构酶参与RNA代谢的研究

STUDIES OF ARABIDOPSIS IMPORTIN PROTEIN AND RNA TOPOISOMERASE IN RNA METABOLISM

杨秋华

11849108

硕士

生物工程

郭红卫

2020-05-30

2020-07-07

哈尔滨工业大学

深圳

RNA代谢过程对于生物的生长发育过程是非常重要的，但是对于RNA代谢过程除了已知的研究成果实际上整个RNA代谢的调控过程还是有很多是我们不知道的。在这里，我们对植物RNA代谢中的RNA降解途径、转录后基因沉默、转录基因沉默三个途径进行研究，试图在拟南芥中找到这三条RNA代谢途径中的其他调控因子。转录基因沉默（TGS）由DCL3切割产生的24-nt siRNA介导，通常与转座子产生的RDR2产物和重复的RNA定向DNA甲基化（RdDM）连锁，其发挥功能同样需要载入AGO4或AGO9中。而有趣的是我们发现该途径的rdr2-2、dcl3-1、ago4-1、ago9-2突变体均可以回复sic1-1突变体的表型，这说明SIC1充当整个TGS途径的调控因子是非常有可能的。此外我们对拟南芥中候选RNA拓扑异构酶AtTop3β也进行了研究，遗憾的是我们发现将Attop3β突变体与PTGS中dcl4-2、ski2-2突变体、RNA降解途径的ein5-1突变体进行杂交后并没有发现表型上的差异。综上，我们通过经典遗传学手段发现SIC1可能参与到RNA代谢的RNA降解、PTGS、TGS三个途径的调控中，但具体机制还需要进一步探索。mRNA的降解对于mRNA的数量和质量控制都是必需的。经典观点认为mRNA仅在其退出蛋白质翻译后才降解，但最近的研究表明mRNA在植物的翻译途径中也有可能边翻译边降解。真核mRNA可以通过两个方向的顺序进行降解：5'-3' 降解，XRN复合体引起的RNA代谢是由几种复杂的外切或内切核糖核酸酶介导的；3'-5' 降解，外切子（exosome）二聚体定义的六聚环。核外切子在进行加工和降解活动需要不同的辅助因子。例如核仁中的MTR4，核质中的HUA ENHANCER2（HEN2）和细胞质中的SUPERKILLER（SKI）复合物。我们发现拟南芥核质转运蛋白SIC1的突变体sic1-1可以被3‘-5’途径中SKI复合体的ski2-2、ski7-1突变体回复其表型。这可能暗示SIC1可以充当3‘-5’途径RNA降解的调控因子。在RNA降解途径发生异常的情况下，那些异常转录物的积累会引起转录后基因沉默，从而带来不利的后果。在拟南芥中已经证明了5'-3'和3'-5'胞质RNA降解途径均充当转基因和内源性PTGS的阻遏物。转录后基因沉默由RDR6/SGS3合成双链RNA触发，随后分别被DCL2、DCL4切割成22-nt siRNA、21-nt siRNA。siRNA可以装入Argonaute（AGO）沉默复合体中，该复合体的目标是同源RNA沉淀和/或翻译抑制。RNA聚合酶可以在主要siRNA靶向后产生次级小干扰RNA（siRNA）分子，从而放大了PTGS的作用。而在我们的研究中发现这一途径仅dcl2-1突变体可以回复核质转运蛋白SIC1的突变体sic1-1的表型。我们猜测可能在PTGS途径中SIC1可能仅通过DCL2参与PTGS过程，但具体机制还需要进一步探索。

The process of RNA metabolism is very important for the growth and development of organisms. However, there are actually many regulatory processes of RNA metabolism that we don’t know. Here, we study the three pathways of RNA metabolism: RNA degradation, post-transcriptional gene silencing, and transcriptional gene silencing. Attempt to find other regulatory factors in these three RNA metabolism pathways in Arabidopsis.The degradation of mRNA is necessary for the quantity and quality control of mRNA. The classic view is that mRNA only degrades after complete protein translation, but recent studies have shown that mRNA may also degrade while translating in plant. Eukaryotic mRNA can be degraded in two directions: 5'-3 'degradation, RNA decay by XRNs complex is mediated by several complex exo- or endo-ribonuclease; 3'-5' Degradation, exosome dimer defined hexamer ring. Exosomes require different cofactors for processing and degradation. For example, MTR4 in nucleolus, HUA ENHANCER2 (HEN2) in nucleoplasm and SUPERKILLER (SKI) complex in cytoplasm. We found that the sic1-1 mutant of the Arabidopsis importin protein SIC1 can be rescued by the ski2-2 and ski7-1 mutants of the SKIs complex in the 3'-5 'pathway. This may imply that SIC1 can act as a regulator of RNA degradation in the 3'-5 'pathway.In the case of abnormal RNA degradation pathways, the accumulation of abnormal transcripts will initiate post-transcriptional gene silencing. It has been demonstrated in Arabidopsis that both 5'-3 'and 3'-5' cytosolic RNA degradation pathways can act as repressors of transgenes and endogenous PTGS. Post-transcriptional gene silencing is triggered by the synthesis of double-stranded RNA by RDR6 / SGS3, which is then cleaved into 22-nt siRNA and 21-nt siRNA by DCL2 and DCL4, respectively. siRNA can loading to the Argonaute (AGO) silencing complex, which targets homologous RNA precipitation and/or translation inhibition. RNA polymerase can generate secondary small interfering RNA (siRNA) molecules after the main siRNA is targeted,IVtherebyamplifying the role of PTGS. In our study, we found that only the dcl2-1 mutant of PTGS can rescued the phenotype of the sic1-1 mutant. We speculate that SIC1 may participate in the PTGS process only through DCL2 in the PTGS pathway, but the specific mechanism needs to be further explored.Transcriptional gene silencing (TGS) is mediated by 24-nt siRNA generated by DCL3 cleavage, and is usually linked to the production of RDR2,which produced by the transposon and repeated RNA directed DNA methylation (RdDM). Its function also needs loading to AGO4 or AGO9. Interestingly, we found that the rdr2-2, dcl3-1, ago4-1, and ago9-2 mutants of this pathway can rescue sic1-1 mutant, which shows that SIC1 acts as a regulator of the entire TGS pathway, possiblely.In addition, we also studied the candidate RNA topoisomerase AtTop3β in Arabidopsis, unfortunately we found that the double mutants of Attop3β mutant cross with dcl4-2, ski2-2 mutant of PTGS pathway and ein5-1 mutant of RNA degradation pathway shows no difference phenotype compared to WT.In summary, we found that SIC1 may be involved in the regulation of RNA metabolism, RNA degradation, PTGS, and TGS through classical genetic methods, but the specific mechanism needs to be further explored.

拟南芥 RNA代谢 核质转运蛋白 RNA拓扑异构酶 AtSIC1 AtTop3β

Arabidopsis RNA metabolism Inportin protein RNA topoisomerase AtSIC1 AtTop3β

中文

联合培养

南方科技大学

杨秋华. 拟南芥核质转运蛋白及RNA拓扑异构酶参与RNA代谢的研究[D]. 深圳. 哈尔滨工业大学,2020.