探討阿拉伯芥中DEAD-box RNA 7解旋酶之功能

Functional characterization of DEAD-box RNA helicase 7, AtRH7, in Arabidopsis

沈育年(Yu-lien Shen)

指導教授 ： 陸重安

國立中央大學/理學院/生命科學系/碩士(2014年)

摘要

在阿拉伯芥中DEAD-box RNA解旋酶依其功能及胺酸序列組成一個蛋白質家族，每個解璇酶蛋白質在RNA代謝過程中扮演著不同的功能，例如: 核醣體生合成、mRNA剪輯、RNA運輸、mRNA轉譯和RNA降解。於此，我們進一步分析一個在阿拉伯芥中被命名為AtRH7/PRH75 RNA解旋酶，首先AtRH7/ PRH75 RNA解旋酶這個基因持續表現於各部位組織，特別是在細胞分化旺盛的組織，同時AtRH7基因亦受到冷逆境所誘導大量表現。在T-DNA突變AtRH7基因的轉植株atrh7-2和atrh7-3具有與突變在核醣體蛋白質相關基因所造成的生長素相關發育(Auxin-relative development)缺陷的相同外表型，此外表型在冷逆境中更為嚴重。然而atrh7的突變株對冷逆境具有不耐受性，原因可能來自於降低CBF1、CBF2和CBF3表現量。在我們的進一步發現，未加工的18S rRNA前驅物在突變株中大量的累積，同時藉由cRT-PCR的實驗發現，其前驅物的累積主要是因為在A2位置剪裁上的缺失所造成的，進一步影響atrh7突變株對一些抑致核醣體小單元的抗生素產生不敏感的外表型，也因此推測AtRH7可能也參與在核醣體的組合過程當中。根據以上的結果，我們的研究表示AtRH7參與rRNA的生合成中A2位置的加工過程及核醣體組合並且在阿拉伯芥適應冷逆境的環境的過程中扮演著很重要的角色。

關鍵字

死盒RNA解旋酶 ； 阿拉伯芥RNA解旋酶7號 ； 核醣體RNA生合成

並列摘要

DEAD box RNA helicases belong to a protein family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation, and RNA decay. This study investigated a DEAD box RNA helicase, AtRH7/PRH75, in Arabidopsis. The AtRH7/PRH75 expression was ubiquitous, was activated in the cell-dividing region, and was induced by cold stress. The phenotypes of 2 allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled the auxin-related developmental defects exhibited in several ribosomal protein mutants and were more severe under cold stress. The atrh7 mutants exhibited decreased CBF1, CBF2, and CBF3 expression, which could be responsible for the subsequent cold intolerance; they also exhibited unprocessed 18S pre-rRNA accumulation. By a circular RT-PCR analysis displayed the accumulated 18S pre-rRNA, indicating that rRNA processing ceased at the A2 site. The atrh7 mutants were hyposensitive to the antibiotics that target to ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. Taken together, our studies indicate that the AtRH7 participates in A2 site processing of rRNA biogenesis and ribosome assembling, and involves in plant development and cold stress tolerance in Arabidopsis.

並列關鍵字

DEAD-box RNA helicase ； AtRH7 ； rRNA biogenesis

參考文獻

Abbasi, N., Kim, H.B., Park, N.I., Kim, H.S., Kim, Y.K., Park, Y.I., and Choi, S.B. (2010). APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis. Plant J. 64, 960-976.

Apirion, D., and Saltzman, L. (1974). Functional interdependence of 50S and 30S ribosomal subunits. Mol. Gen. Genet. 135, 11-18.

Asakura, Y., Galarneau, E., Watkins, K.P., Barkan, A., and van Wijk, K.J. (2012). Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis. Plant Physiol. 159, 961-974.

Aubourg, S., Kreis, M., and Lecharny, A. (1999). The DEAD box RNA helicase family in Arabidopsis thaliana. Nucleic Acids Res. 27, 628-636.

Axt, K., French, S.L., Beyer, A.L., and Tollervey, D. (2014). Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage. PLoS One 9, e85703.