阿拉伯芥細胞核編碼的一個粒線體CRM結構域蛋白CFM6之功能性分析

Functional characterization of CFM6, a nuclear-encoded mitochondrial CRM domain-containing protein, in Arabidopsis thaliana

林威至(Wei-Chih Lin)

指導教授 ： 鄭萬興

共同指導教授 ： 張英峯(Ing-Feng Chang)

國立臺灣大學/生命科學院/植物科學研究所/博士(2021年)

https://doi.org/10.6342/NTU202104481

摘要

粒線體是由原核細菌(α-proteobacteria)演化而來，在長期互利共生的關係下，逐漸演化成真核細胞的能量工廠並轉移了多數的基因到核基因組(nuclear genome)。因此，維持粒線體的呼吸作用正常運作並產生能量，需要核基因所編碼的蛋白質對粒線體基因進行多種後轉錄修飾。特別是許多組成粒線體呼吸傳遞鏈的蛋白質，這些編碼蛋白質的基因帶有退化的Group II 內含子，己失去自行剪切的能力，需要協同多種細胞核的蛋白質來完成內含子剪切(intron splicing)。CRM蛋白家族是細胞核編碼的核酸結合蛋白，目前多數的研究報導CRM蛋白經由參與group I 和group II內含子剪切來調控葉綠體基因的表現。然而CRM蛋白在粒線體中扮演的生理功能及作用機制仍未完全明瞭。本研究進一步分析阿拉伯芥CFM6基因的生理功能及分子機制，並揭開這個具單一CRM蛋白結構域(CRM domain)的家族成員在粒線體中扮演的角色。當T-DNA插入造成CFM6基因突變，會嚴重影響cfm6突變株的發育，包含生長遲緩、葉片捲曲、種子皺縮、延遲胚及花粉的發育。實驗結果顯示CFM6蛋白具專一性調控粒線體nad5基因第四個內含子的剪切；因此，在cfm6突變株中造成未經剪切的轉錄物(pretranscripts)。於cfm6突變株中大量表現CFM6-YFP（CFM6蛋白融合黃色螢光蛋白）可恢復植株發育不良的性狀及內含子剪切功能的缺失。因nad5蛋白為組成呼吸傳遞鏈複合體 I (respiratory complex I) 的元件，其轉錄後內含子剪切的缺失進一步造成cfm6突變株粒線體內複合體 I 的生合成降低及活性損壞，伴隨粒線體形態發育異常，並且誘導替代呼吸途徑(alternative respiration pathway)相關基因的表現。根據RNA-seq分析的結果，CFM6基因突變擾動細胞核及胞器基因之表現，全數2764個受擾動的基因中，多數與光合作用相關的基因表量下降，而與核糖體生合成相關的基因表現量則呈現上升的趨勢。目前的研究結果顯示CFM6是一個參與粒線體內nad5基因第四個內含子剪切的剪切因子，在粒線體呼吸作用複合體 I 的生合成及植物生長發育過程中扮演重要的角色。

關鍵字

阿拉伯芥 ； CRM蛋白結構域 ； 胚胎發生 ； Group II內含子 ； 雄不稔 ； 粒線體NADH去氫酶

並列摘要

Mitochondria derived from α-proteobacteria by endosymbiosis act as the cellular powerhouse in eukaryotic host cells. Most genes involved in regulation of mitochondrial gene expression were transferred to the nuclear genome during evolution. Therefore, functional respiration demands the various posttranscriptional regulation from nuclear-encoded proteins, particular in the RNA splicing factors. Many mitochondrial genes encoding components of the respiratory chain are interrupted by group II introns, which have lost the ability to self-splice and require numerous nuclear-encoded proteins as cofactors. Chloroplast RNA splicing and ribosome Maturation (CRM) domain is an RNA-binding domain, and CRM domain-containing proteins regulate chloroplast gene expression via splicing manipulation of the group I and group II introns. However, their functions in mitochondria are less understood. In this study, the biological and molecular functions of Arabidopsis CFM6, a protein with single CRM domain, were characterized. The null T-DNA mutants of cfm6 exhibited severe growth defects, including stunted growth, curled leaves, shrunken seeds, delayed embryogenesis and pollen development. CFM6 functions specifically in the splicing of intron 4 of mitochondrial nad5, which encodes a subunit of respiratory complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in the cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in nad5 also greatly reduced complex I activity and biogenesis, together with an abnormal mitochondrial morphology in cfm6 mutants. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Besides, the CFM6-mediated alteration of nuclear and organellar transcriptome were revealed by RNA sequencing. Gene ontology (Go) analysis of all 2764 differentially expressed genes indicated that photosynthesis-related genes were down-regulated, while the genes associated with ribosome biogenesis were up-regulated in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

並列關鍵字

Arabidopsis thaliana ； CRM domain-containing protein ； Embryogenesis ； Group II intron ； Male sterility ； Mitochondrial respiratory complex I

參考文獻

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