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Fig. 3 | Epigenetics & Chromatin

Fig. 3

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

Fig. 3

EK transcriptional activation affects CMT1 downstream expression. a Schematic representation of the CMT1 gene and the insertion of EK within exon 13. CMT1 exons are numbered and shown as blue boxes and introns as black lines. Primers used to amplify upstream and downstream CMT1 sequences from cDNA are marked by arrowheads and numbered (P1–P7). The red broken line indicates the splice acceptor site of exon 14 (based on 27). EK LTRs are marked by red arrowheads. b CMT1 upstream transcript is alternatively spliced showing intron 12 retention. cDNA was prepared from RNA extracted from flowers derived from WT Col, WT Ler, ddm1, kyp2, cmt3, ago4 and rdr2 and subjected to PCR to amplify CMT1 RNA sequences using primer set 1 + 2 (ex10-F + ex11-R) or primer set 3 + 4 (ex11-F + ex13-R). Note that primer set 3 + 4 yielded two PCR fragments SP1 and SP2. Actin was used as a reference. PCRs were performed for 35 cycles except for actin (30 cycles). M indicates DNA molecular size markers. The predicted alternative spliced variants SP1 and SP2 are schematically shown below. c CMT1 downstream expression using primer set 5 + 6 (ex14-F + ex16-R). WT Ler gDNA was used as a reference for PCR product derived from RNA. Note the enhanced downstream CMT1 expression in kyp2 and cmt3 mutants. Actin was used as a reference. Ler genomic DNA (gDNA) was used to confirm amplification from RNA. PCRs were for 35 cycles except for actin (30 cycles). d Analysis of splicing out of the entire EK retroelement using primer set 6 + 7 (ex13-F + ex16-R) on both sides of the EK insertion site. Note lack of a PCR product in kyp2 and cmt3 mutant. Actin was used as a reference RNA. Col genomic DNA (gDNA) was used to confirm amplification from RNA. PCR was for 42 cycles except for actin (30 cycles)

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