CMT3 and SUVH4/KYP silence the exonic retroelement Evelknievel to allow for reconstitution of CMT1 mRNA

Abstract

Highlights: O_LISilencing of the intragenic Evelknievel (EK) retroelement is maintained by CMT3-KYP/SUVH4 independently of DDM1 and the RdDM pathways.\nC_LIO_LIMethylation and silencing of EK is required for transcription through the EK retroelement.\nC_LIO_LISilencing of EK allows for splicing out of the entire EK and reconstitution of an intact CMT1 mRNA.\nC_LI\n\nBackgroundCHROMOMEHYLASE1 (CMT1) has long been considered a non-essential gene because, in certain Arabidopsis ecotypes, the CMT1 gene is disrupted by the retroelement Evelknievel (EK), inserted within exon 13, or contains frame-shift mutations resulting in a truncated, non-functional protein. Here, we wanted to explore the regulatory pathway responsible for EK silencing in the Ler ecotype and its effect on CMT1 transcription.\n\nResultsMethylome databases confirmed that EK retroelement is heavily methylated but 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..\n\nConclusionsOur 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 a very low level of an intact, functional mRNA and possibly to retrieval of an active protein.