French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, 84990, Midreshet Ben Gurion, Israel.
The School of Plant Sciences and Food Security, Tel-Aviv University, 69978, Tel Aviv, Israel.
Epigenetics Chromatin. 2018 Nov 16;11(1):69. doi: 10.1186/s13072-018-0240-y.
The Chromomethylase 1 (CMT1) has long been considered a nonessential gene because, in certain Arabidopsis ecotypes, the CMT1 gene is disrupted by the Evelknievel (EK) retroelement, inserted within exon 13, or contains frameshift mutations, resulting in a truncated, non-functional protein. In contrast to other transposable elements, no transcriptional activation of EK was observed under stress conditions (e.g., protoplasting).
We wanted to explore the regulatory pathway responsible for EK silencing in the Ler ecotype and its effect on CMT1 transcription. Methylome databases confirmed that EK retroelement is heavily methylated and methylation is extended toward CMT1 downstream region. Strong transcriptional activation of EK accompanied by significant reduction in non-CG methylation was found in cmt3 and kyp2, but not in ddm1 or RdDM mutants. EK activation in cmt3 and kyp2 did not interfere with upstream CMT1 expression but abolish transcription through the EK. We identified, in wild-type Ler, three spliced variants in which the entire EK is spliced out; one variant (25% of splicing incidents) facilitates proper reconstitution of an intact CMT1 mRNA. We could recover very low amount of the full-length CMT1 mRNA from WT Ler and Col, but not from cmt3 mutant.
Our findings highlight CMT3-SUVH4/KYP as the major pathway silencing the intragenic EK via inducing non-CG methylation. Furthermore, retroelement insertion within exons (e.g., CMT1) may not lead to a complete abolishment of the gene product when the element is kept silent. Rather the element can be spliced out to bring about reconstruction of an intact, functional mRNA and possibly retrieval of an active protein.
长期以来，色氨酸甲基转移酶 1（CMT1）被认为是一个非必需基因，因为在某些拟南芥生态型中，CMT1 基因被 Evelknievel（EK）反转元件破坏，插入外显子 13 内，或包含移码突变，导致截短的、无功能的蛋白质。与其他转座元件不同，在胁迫条件下（例如原生质体化）没有观察到 EK 的转录激活。
我们希望探索负责 Ler 生态型中 EK 沉默的调控途径及其对 CMT1 转录的影响。甲基化组数据库证实，EK 反转元件高度甲基化，甲基化向 CMT1 下游区域延伸。在 cmt3 和 kyp2 中发现 EK 强烈转录激活伴随着非 CG 甲基化的显著减少，但在 ddm1 或 RdDM 突变体中没有发现。cmt3 和 kyp2 中的 EK 激活不会干扰上游 CMT1 表达，但通过 EK 阻止转录。在野生型 Ler 中，我们鉴定出三种剪接变体，其中整个 EK 被剪接掉；一种变体（25%的剪接事件）有助于正确重建完整的 CMT1 mRNA。我们可以从 WT Ler 和 Col 中回收非常少量的全长 CMT1 mRNA，但不能从 cmt3 突变体中回收。
我们的研究结果强调了 CMT3-SUVH4/KYP 作为主要途径，通过诱导非 CG 甲基化来沉默内含子中的 EK。此外，当元件保持沉默时，元件插入外显子（例如 CMT1）不一定会导致基因产物的完全消除。相反，元件可以被剪接掉，从而重建完整的、功能性的 mRNA，并可能恢复活性蛋白。
