The Arabidopsis DCP2 gene is required for proper mRNA turnover and prevents transgene silencing in Arabidopsis
Article first published online: 30 AUG 2012
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 72, Issue 3, pages 368–377, November 2012
How to Cite
Thran, M., Link, K. and Sonnewald, U. (2012), The Arabidopsis DCP2 gene is required for proper mRNA turnover and prevents transgene silencing in Arabidopsis. The Plant Journal, 72: 368–377. doi: 10.1111/j.1365-313X.2012.05066.x
- Issue published online: 29 OCT 2012
- Article first published online: 30 AUG 2012
- Accepted manuscript online: 28 MAY 2012 10:12AM EST
- Received 11 April 2012; revised 16 May 2012; accepted 23 May 2012; published online 30 August 2012.
File S1. Overview on the forward genetic screen.
File S2. Map based cloning of the ITS1 locus.
File S3. List of all Oligos used in this study.
Figure S1. MP17:GFP expression incotyledons of its1/MP17:GFP. Silencing ofMP17:GFP can not be observed in cotyledons. Confocal imagesshow epidermal cells of cotyledons stained with propidium iodide ofCol-0, Col-16 and its1/MP17:GFP. Bars represent 20µm.
Figure S2. dcp2-1 cannot complement theits1 silencing phenotype. (a) The organization ofDCP2 including exons and introns was modified based onGoeres et al., (2007). Light grey boxes represent the DCP2domain, whereas dark grey boxes represent the Nudix domain. Thedcp2-1 T-DNA is located within exon three, whereas theits1 mutation is located in exon four. (b) For genotypingthe F1 individuals of a cross between its1 anddcp2-1, genomic DNA was extracted and analyzed by PCR usingT-DNA specific primers. All individuals containing both oneits1 allele and an additional dcp2-1 T-DNA allele displayed a loss of MP17:GFP fluorescence in source leaves as shown in (c).
Figure S3. Genotypic analysis ofits1/MP17:GFP and confirmation of complementation. (a) Sincethe C to T exchange in AtDCP2 deletes a restriction site forSacII, this cleaved amplified length polymorphism (CAPS) allowsgenotyping of the its1 mutation. To this end, a 427 bp longPCR fragment specific for the CAPS polymorphism in DCP2 wasamplified and subsequently digested using the SacII restrictionenzyme. A homozygous its1 mutation yields a non cleavablePCR fragment whereas heterozygous mutation will give a partial andthe wild-type a full digest of the PCR fragment. (b) Analysis oftransformation in basta resistant plants. Six basta resistantcandidates were analyzed. Genomic DNA was extracted and theits1 genotype was determined according to (a). All testedcandidates where heterozygous indicating an additional wild-typeallele within the its1 mutant background. (c) To excludecontaminations we also determined the genotype of the endogenousits1 mutation. Thus, it was necessary to amplify a fragmentspecific for the endogenous locus, which was not present on theT-DNA used for complementation. This fragment was purified andfurther used as a template for analysis according to (a). Allindividuals were homozygous in the its1 mutation. Thus, allsix tested individuals were its1 mutants successfullytransformed with a wild-type allele of DCP2.
Figure S4. Phenotypic analysis ofits1/MP17:GFP complementation. (a) For analysis ofcomplementation six basta resistant individuals were analyzed byconfocal laser scanning microscopy, five weeks after bastatreatment. All individuals displayed MP17:GFP fluorescence in allsource leaves at any developmental stage. Thus, no silencing wasdetectable which clearly shows that wild-type DCP2 cancomplement the its1 silencing phenotype. (b) Northern blotanalysis of its1 complementation including Col-0, Col-16,its1/MP17:GFP and its1/MP17:GFP transformedwith wild-type DCP2. 18S rRNA probe was used as a loading control.
Figure S5. Measurement of uncapped mRNA usingqSL-RT-PCR. (a) 5’ RACE PCR of the MP17:GFPtranscript. The entire 5’ sequence of the MP17:GFPmRNA was determined using Clontech’s SMARTer RACE cDNAAmplification Kit. Ten clones were sequenced and aligned to theCaMV35S:MP17:GFP sequence using the Geneious software. Foralignment the SMART extension and the SMARTer oligonucleotide weretaken into account. (b) Design of the DNA splint. The sequence ofthe DNA splint which anneals to both, the RNA anchor and the5’ UTR of MP17:GFP was designed as describedpreviously (Blewett et al., 2011). The MP17:GFPsplint sequence shown is reverse complementary to the DNA splint,which anneals to the RNA anchor and the 5’ UTR ofMP17:GFP mRNA. (c) Principle of splinted ligation qRT-PCR.For qSL-RT-PCR analysis total RNA containing capped and uncappedMP17:GFP mRNA was extracted and subjected to splintedligation. In a concerted reaction the DNA splint annealed to theuncapped 5’ UTR of MP17:GFP and to the RNA anchor. TheRNA anchor and the 5’UTR were ligated following DNAse digestof the DNA splint. The artificial extended RNA molecule wasdetected by qRT PCR using RNA anchor specific primers. Thisanalysis allows the detection of only uncapped full length mRNA.Orange arrow indicates the RNA anchor specific primer. Green arrowsindicate gene specific primers.
Figure S6. Silencing of MP17:GFP inits1 is RDR6 dependent. Confocal images show epidermal cellsof source leaves stained with propidium iodide of Col-0, Col-16,its1/MP17:GFP and its1/MP17:GFP/sgs2-1. Bars represent 20 µm.
Figure S7. The BAR gene inits1/eGFP remains unaffected of PTGS. (a) Analysis ofCol-0, DCP2/eGFP and its1/eGFP one dayfollowing basta treatment. (b) Northern blot analysis of Col-0,DCP2/eGFP and its1/eGFP. BAR mRNA isdetectable. 18S rRNA probe was used as a loadingcontrol. (c) Same samples as in (b) were subjectedto small RNA blot analysis. 5S rRNA was used as a loading control.
Figure S8. Structural analysis of the DCP2protein. (a) Modelling of the AtDCP2 protein. The structure ofSchizosaccharomyces pombe (She et al., 2006) was usedto create a homology model of AtDCP2. Glutamate residues 154, 157and 158 predicted for catalysis are shown in yellow. Lysine residue243 and 248 predicted for RNA binding are shown in cyanine. Theasparagine residue 138 and lysine residue 140 which was shown to beimportant for decapping activity are shown in cyanine. (b)Alignment of wild-type DCP2 to its1 DCP2. Box shade serverwas used to align wild-type and its1 DCP2. Residues shown in (a) are highlighted with asterisks.
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