Intron distance to transcription start (nt)*

Transcription

1 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 1 Supplemental Table 1. Characteristics of introns used in this study Intron name Length intron (nt) Length cloned fragment (nt) AT content cloned fragment (%) U content intron (%) Intron distance to transcription start (nt)* Intron distance to closest Dicer cut (nt) * Endogenous host gene Identifier or accession number i Ath-MIR163 At1g66725 ichs Phy-chsA X igus ST-LS1 X i172b Ath-MIR172b At5g04275 i172b Ath-MIR172b At5g04275 iubq Ath-UBQ10 At4g05320 itrp Ath-TRP1 At5g17990 itch Ath-TCH3 At2g41100 i172a 1 st Ath-MIR172a At2g28056 i172a 2 nd Ath-MIR172a At2g28056 GFP mgfp5-er U Engineered 3 introns were inserted downstream of the intron-less variant, at position 634 of the MIR163 construct. 5 introns right in front of the start. * Indicates the distance to the transcription start/ closest Dicer cut in the endogenous host gene. Abbreviations: Ath Arabidopsis thaliana, ST Solanum tuberosum, Phy Petunia x hybrid.

2 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 2 Supplementary Table 2: IMEter scores Scores were calculated at: for version 1 (v.1) for version 2 (v.2) Only intron sequences (see Supplemental Table 3), not surrounding nucleotides in cloned fragments were used. Intron Name IMEter score v.1 IMEter score v.2 iubq itrp itch i i172a first intron i172a second intron i172b 5 intron

3 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 3 Supplemental Table 3. Sequences of introns and cloned fragments used in this study Intron Name i163 ichs igus iubq itrp1 itch3 i172b 5 i172b 3 GFP i172a 1 st i172a 2 nd Sequence of cloned fragment, intron sequence underlined ggttgtgcctgaaaatatgggtttctaggtttaattgatgaaaagagataacgtaagtcatgcacatgtaattaaattcatgcccctaactattaattcttgcaagtaattaaaaaataaaacgtacacaaaatatatttatgatcactaatcctttttaactactaa cattaacgatttgatctatcaatgtctaatgatttcagggaaattttttgtttcctcatgtttacacgtgtcaacatcctccgaaggaatgcaaatggttgtggaaaaaaaaaaaaaaaaaagaatgcaaatggttgagctcgtgttgggctttgttttcttatttt aggtgtaagaatttcttatgttacattattacattcaacgttttatcttaattggctcttcatttgattgaaatttgacaattatttcttgtttttttttttgtcacactctttttgggttggggtggccgacgaattgtgggaaggtagaaagaggggaggacttttgttatactcc attagtaattactgtttccgtttcaatttatgtgacaatatttcctttttagtcggttccaaaagaaaatgtcagcattataaacaatttaattttgaaattacaattttgccattaataaaatgatttacaaccacaaaagtatctatgagcctgtttgggtgggcttataa gcagcttattttaagtggcttataagtcaaaaagtgacantttttgagaagttagaaaatcctaacttctcaaaaagtagcttttaagccacttatgacttataagtccaaaaatttttaagttaccaaacatatattaatgggtttataagcttataagccacttttaa gctcacccaaacgggttctatgtctcactttagactacaaattttaaaagtcttcatttatttcttaatctccgtggcgagtnaaactataacacataaagtgaaacggagggaataagatggagtcataaactaatccaaatctatactctctccgttaatttgtttt ttagtttgatttggtacattaataaaacagatttttcgaaggttataaacacagacagatgtttcccagcgagctagcaaaattccaagatttctgtcgaaaattcgtgtgtttctagctagtacttgatgttatctttaaccttttagtaattttttgtccttttctttctatttttc atcttacaatgaattatgagcaagttccttaagtagcatcacacgtgagatgttttttatgatattgactaaatccaatctttaccattccttaactagtaaaatacaacacatgttaattgatacattgcttaacactgaggttagaaaattttagaaattagttgtcca aatgctttgaaattagaaatctttaatcccttatttttttttaaaatgttttttctcactccaaagaaagagaaactgacatgaaagctcaaaagatcatgaatcttactaactttgtggaactaaatgtacatcagaatgtttctgacatgtgaaaatgaaagctctta attttcttcttttatttattgagggtttttgcatgctatgcattcaatttgagtactttaaagcacctataaacacttacttacacttgccttggagtttatgttttagtgttttcttcacatcttttttggtcaatttgcaggt tggtatcagcgcgaagtctttataccgaaaggttgggcaggccagcgtatcgtgctgcgtttcgatgcggtcactcattacggcaaagtgtgggtcaataatcaggaagtgatggagcatcagggcggctatacgccatttgaagccgatgtcacgccgt atgttattgccgggaaaagtctacgtaagtttctgcttctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttcaaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttataactt ttctaatatatgaccaaaatttgttgatgtgcaggtatcaccgtttgtgtgaacaacgaactgaactggcagactatcccgccgggaatggtgattaccgacgaaaac ttctcaaaatcttaaaaactttctctcaattctctctaccgtgatcaaggtaaatttctgtgttccttattctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtatatgttctttggtttagattctgttaatcttagatcgaagacgattttctgggtttgatcgttag atatcatcttaattctcgattagggtttcatagatatcatccgatttgttcaaataatttgagttttgtcgaataattactcttcgatttgtgatttctatctagatctggtgttagtttctagtttgtgcgatcgaatttgtcgattaatctgagtttttctgattaacagatgca GATCTTTGTTAAGACTCTCACCGGAAAGACAATCACCCTCGAGGTGGAAAGCTCCGACACCATCGACAACGTTAAGGCCAAGATCCAGGATAAGGAGGGCATTCCTCCGG ATCAGCAGAGGCTTATTTTCGCCGGCAAGCAGCTAGAGGATGGCCGTACG CCAGTAGTTGCGGAAGAAGCAACTTGACCGGAGTTAGAATTTTTCCGACATTGAGTCGGCGACGATTCTCATCGATCGGAGCTGTTTCCCCAATTCGTGGGGATGCTCAA TCTTCCTTTAGTCGCAGgtaaagcctcgatttttgggtttaggtgtctgcttattagagtaaaaacacatcctttgaaattgtttgtggtcatttgattgtgctcttgatccattgaattgctgcagCTCTTTCGCTTGCTCTCAGAATCTC GG Ggtatggttcatcaactctttccatttaatcgtaatgttggatcatgatcatcttcaaagcaatgaaatgactaacacaagtccttgattactttttgtag ccatgcattcgcttatagtttcacacaatttgttttcttcacggtctaaaatcagaagattccatatattttcttatgacgtaaaaggaccacttataagttgacacgtcagcccttggattcgtgaggtttttctctctacttcacctatctacttttcctcatatcccactgc ttttctccttcttgttcttgtttttctcgtttttttcttcttcttctccaagaaaatagagatcgaaaagattagatctattttgtgtagcaagaaattatcattttcgtttcttcattcatatattgttctattatgttgtacaataatagatactcgatctcttgtgcgtgcgtaaattttat acaagttgtcggcggat aagcttgagaaagaaacttgaagatattgattgaagtctggatcgatctttggtaaatctctctcttgattagttttaagaatcacttttttttttctgtgtttgaacatgtttacatatatcatctatgtctcaatatatatattttcttaatctagggtcaatgacggattaggg cgttaattacaatgaatatggaaaaactattttgcctttgatcttgacttgagtgttgatgaacagatgtataatgttatgtagtatgtactgtattttttctagaatcattctttagtctccaactctccattaatcaaatgaggtccttataggtaatgctatgatcaagaa caacaagatcgtgagcacagatcggccagttcggtcactttttaaaagcggccgccaccgcggtgg atgaagactaatctttttctctttctcatcttttcacttctcctatcattatcctcggccgaattcagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatg caacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaagcggcacgacttcttcaagagcgccatgcctgagggata cgtgcaggagaggaccatcttcttcaaggacgacgggaactacaagacacgtgctgaagtcaagtttgagggagacaccctcgtcaacaggatcgagcttaagggaatcgatttcaaggaggacggaaacatcctcggccacaagttggaataca actacaactcccacaacgtatacatcatggccgacaagcaaaagaacggcatcaaagccaacttcaagacccgccacaacatcgaagacggcggcgtgcaactcgctgatcattatcaacaaaatactccaattggcgatggccctgtccttttacc agacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaacacgacgaactctaa gtaggtttgtttctacttgaagtttttttttttcacctttatgttaacatatcctcccgtttatttttatttgttatacataaagatctgacaaagaacttttgtgggtatcttgtttcatgtgataacattgagcatttgatctcag gtaagagagatctacttaaggtaactaatatatatgccttatttcttatgttgccaatcccaccttgcatttccaaaacaacacaatatagttttgttttgttagaaacaaaaaaggatattatatatggaagttgagataatatatataacaggtctatatggaacta ttgttgtctagcaaagaaaaggttttcaagacacactgtcagttctttgttttgttggttcatgttggctattacattcaaattgagaatcaatcaagaacttgttgggtggaccttacacacattgtaggctaggcatattcagtccaacctatttgtgtcccctctattta catatttagatttcgtccttataactgatattgaaacaatgaagaatatatactctctttttctctttattctgttataagggaacaataaatctatagcatcatatatactcagactgtgaacaaaacctattcacagttatgagtttcatgttttgtttatgagcacaacct cccaaacttgtttccaaaacaaattctataaatattagtattatttttggttattatgtcttgtagtacatcttgttatatgtgtgtttagtaatgttttctatgttatggagatgtgtgtgtctaaattactttccattagtcattgtagaaataaatctttttttttttatcatag

4 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 4 Supplemental Table 4: Oligonucleotides Restriction sites in oligonucleotides are in capital letters. Capitalized and bold letters indicate nucleotides that were altered relative to the template. A description of final plasmids is given in Supplemental Table 5. Abbreviations: gdna, genomic DNA from ecotype Col-0; cdna, cdna from ecotype Col-0, dcl1 mutants or transgenic lines. Locus Purpose PCR Template Sequence 5 to 3 (restriction sites in capitals) MIR163 cloning 163-1, gdna gtaccttagataaaccgaccaaaacccgg MIR163 cloning gdna aaaataagaaaacaaagcccaac MIR163 cloning gdna ttttgtgtacgttttattttttaattacttgc MIR163 cloning 163-3, gdna, cdna gatcatgtccactactcaagagc MIR163 cloning 163-3, gdna, cdna aaaataagaaaacaaagcccaac MIR163 cloning side GGATCCggttgtgcctgaaaatatgggtttc MIR163 cloning side TCTAGAaaaataagaaaacaaagcccaac MIR163 cloning side CTCGAGggttgtgcctgaaaatatgggtttc MIR163 cloning side GTCGACaaaataagaaaacaaagcccaac MIR163 cloning 163Δ gagataaccaaactcatgcacatctaatta MIR163 cloning 163Δ taattagatgtgcatgagtttggttatctc MIR163 RT-PCR stemloop cdna gtaccttagataaaccgaccaaaacccgg MIR163 RT-PCR stemloop cdna caacctatagagaaactggacaaaacacg MIR163 RT-PCR intron cdna ggatccggttgtgcctgaaaatatgggtttc MIR163 RT-PCR intron cdna cacaaccatttgcattccttcggagg mir163 qrt-pcr cdna ccgacgttgaagaggacttggaac mir163 qrt-pcr cdna gtcgtatccagtgcagggtccgaggtattcgcactggatacg acatcgaa mir163 Northern - atcgaagttccaagtcctcttcaa mir172 Northern - atgcagcatcatcaagattct mir172a qrt-pcr cdna ccgtcgagaatcttgatgatg

5 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 5 mir172a qrt-pcr cdna gtcgtatccagtgcagggtccgaggtattcgcactggatacg acatgcag MIR172a cloning 172a-1, 172a-2 gdna, cdna aaaaatggaagactaatttccgga MIR172a cloning 172a-1, 172a-2, 172a-3, 172a-4 gdna, cdna tgaggtacttattttcgtaaaacaac MIR172a cloning 172a-3, 172a-4 gdna, cdna gcttgtggatctattaatgtcttg MIR172a cloning 172aΔ 172a-3 cagatcttcttcaccaagctttgtttctacttg MIR172a cloning 172aΔ 172a-3 caagtagaaacaaagcttggtgaagaagatctg MIR172a RT-PCR intron (first intron) cdna cctctctctctctctccctctgt MIR172a RT-PCR intron (first intron) cdna gcttgtggatctattaatgtcttg MIR172b cloning 172b-1, 172b-2, 172b-3 gdna, cdna GAATTCactagtcttgtgtgcacccattt MIR172b cloning 172b-1 gdna tgtctggtctctggacgaactat MIR172b cloning 172b-2, 172b-3 gdna, cdna TCTAGAaagatcgatccagacttcaatca MIR172b RT-PCR 5 intron cdna ttagtttgttgtgtgtgagactttg MIR172b RT-PCR 5 intron cdna gatccgccgacaacttgtat MIR172b RT-PCR 3 intron cdna attgaagtctggatcgatctttg MIR172b RT-PCR 3 intron cdna tgtctggtctctggacgaactat MIR172b cloning i172b5 gdna GGATCCccatgcattcgcttatagtttca MIR172b cloning i172b5 gdna TCTAGAatccgccgacaacttgtat MIR172b cloning i172b3 gdna GGATCCaagcttgagaaagaaacttgaaga MIR172b cloning i172b3 gdna GCGGCCGCttttaaaaagtgaccgaactgg MIR173 cloning gdna GAATTCtcatcgtaatctcagcccaatac MIR173 cloning gdna GGATCCccgaatctccaaattgaaataca MIR173 RT-PCR cdna gaattctcatcgtaatctcagcccaatac MIR173 RT-PCR cdna ggatccccgaatctccaaattgaaataca mir173 Northern - gtgatttctctctgcaagcgaa chsa cloning side pfgc5941 GGATCCaggtgtaagaatttcttatgttacattattac (chromdb.org) chsa cloning side pfgc5941 TCTAGAacctgcaaattgaccaaaaaagatgtgaag (chromdb.org) chsa cloning side pfgc5941 (chromdb.org) CTCGAGgtgtaagaatttcttatgttacattattac

6 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 6 chsa cloning side pfgc5941 GTCGACacctgcaaattgaccaaaaaagatgtgaag (chromdb.org) GFP cloning GFP@3 side mgfp5 GGATCCatgaagactaatctttttctctttct containing plasmid GFP cloning GFP@3 side mgfp5 TCTAGAttagagttcgtcgtgtttgtatagttc containing plasmid GFP cloning GFP@5 side mgfp5 CTCGAGatgaagactaatctttttctctttct containing plasmid GFP cloning GFP@5 side mgfp5 AAGCTTttagagttcgtcgtgtttgtatagttc containing plasmid GUS cloning side plasmid GGATCCtggtatcagcgcgaagtctttat containing GUS with intron [1] GUS cloning side plasmid TCTAGAgttttcgtcggtaatcaccattc containing GUS with intron [1] GUS cloning side plasmid CTCGAGtggtatcagcgcgaagtctttat containing GUS with intron [1] GUS cloning side plasmid GAATTCgttttcgtcggtaatcaccattc containing GUS with intron [1] TCH3 cloning itch3 gdna GGATCCggtatggttcatcaactc TCH3 cloning itch3 gdna TCTAGActacaaaaagtaatcaag Terminator RT-PCR 35S Terminator cdna ctagagtccgcaaaaatcaccagtc Terminator RT-PCR 35S Terminator cdna catactaagggtttcttatatgctcaacaca TRP1 cloning itrp1 gdna GGATCCccagtagttgcggaagaagc

7 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 7 TRP1 cloning itrp1 gdna TCTAGAccgagattctgagagcaagc Tubulin RT-PCR cdna gagccttacaacgctactctgtctgtc At5g62690 Tubulin RT-PCR cdna acaccagacatagtagcagaaatcaag At5g62690 U6 Northern - AGGGGCCATGCTAATCTTCTC UBQ10 cloning iubq gdna GGATCCttctcaaaatcttaaaaactt UBQ10 cloning iubq gdna ACTAGTcgtacggccatcctctag SALK_ genotyping gdna caattttgttcgtgtgtggtg SALK_ genotyping gdna aggtacttgtcttccaccaatca LBb1.3 genotyping gdna attttgccgatttcggaac mirna qrt-pcr universal primer cdna gtgcagggtccgaggt

8 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 8 Supplemental Table 5: Alternative binary names Transgenes were PCR amplified with Phusion Taq (Finnzymes), gel purified (QIAquick, Qiagen; GeneJet, Fermentas), ligated into pbluescript SK- (pbsk; Strategene) that had been linearized with SmaI (Fermentas), and sequence-verified. Details on oligonucleotides and PCR templates are listed in Supplementary Table 4. Transgenes were shuttled into a home-made Gateway entry vector pjlblue (pbsk multiple cloning site between AttL1, AttL2 sites) using restriction sites introduced through oligos (capitalized in Supplementary Table X) or sites available in the pbsk multiple cloning site. Extra introns and GFP were added to the fragment already present in the entry vector using restriction sites available in the plasmid. Final products were recombined into pb2gw7 [2], which contains a 35S promoter and a 35S terminator, using LR clonase (Invitrogen). Final binary plasmids were shuttled into Agrobacterium strain GV3101. Abbreviation used in paper Description Alternative name prs MIR163 variant with intron MIR163 variant ending in intron MIR163 variant with intron MIR163 variant, intron omitted Δ MIR163 variant with mutated splice donor site a-1 Long MIR172a variant with two introns a-2 Long MIR172a variant, both introns omitted a-3 Short MIR172a variant with intron a-4 Short MIR172a variant, intron omitted aΔ Short MIR172a variant with mutated splice donor site b-1 Long MIR172b variant with 5 and 3 intron b-2 MIR172b variant with 5 intron 2281

9 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 9 172b-3 MIR172b variant, 5 intron omitted MIR173 fragment 2420 i plus 3 i i plus 5 i ichs plus 3 ichs 2414 ichs plus 5 ichs 2534 igus plus 3 igus 2636 igus plus 5 igus 2638 i172b plus 3 i172b5 (5 intron of MIR172b) 2635 i172b plus 3 i172b3 (3 intron of MIR172b) 25R iubq plus 3 iubq U1 itrp plus 3 itrp1 T8 itch plus 3 itch3 9.3 GFP plus 3 GFP plus 5 GFP plus 5 GFP 2520

10 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 10 Supplemental Figure 1 Comparison of intron ability to increase mirna accumulation and various traits listed in Supplemental Table 1. MiRNA levels (left axis in all graphs) were approximated from northern blots in transient assays with transgenes in which introns had been added at the 3 side of the intron-less construct. The MIR163 intron, which produced the highest levels, was used as a reference point and set to 100%. A. Intron length; B. Length of the cloned fragment; C. AT content of the cloned fragment; D. U content of the intron. A B C D

11 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 11 Supplemental Figure 2. Precursor RT-PCRs A. RT-PCR on transgenic plants carrying the different MIR163 transgenes (background MIR163 KO line). The different oligonucleotides are indicated on the top, basta refers to the resistance gene on the binary plasmid, (*) indicates an unrelated transgenic plant that was used as a control. B. RT-PCR referring to Fig. 3A: Transgenes are indicated on top, and correspond to and 163-4, but carry an additional GFP coding sequence at the 5 side. Northern blots (middle) indicate mature mirna accumulation in plants that were segregating as wild-type looking from a dcl1-7 heterozygous population ( seg ). RT-PCR with different oligonucleotides (bottom) was performed on the same RNA as the northern blot.

12 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 12 Supplemental Figure 3. Cartoon Model Proposed model of 3 intron action: In the presence of a 3 intron, elements within the intron enhance processing of mirna precursor RNAs or stability of mature mirnas In the absence of dicing, splicing is more effective, while mature mirna accumulation is reduced when no 3 intron is present.

13 Schwab et al. SOM: Enhanced microrna accumulation through stemloop-adjacent introns 13 Supplemental References 1. Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an introncontaining marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacteriummediated plant transformation. Mol Gen Genet 220: Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:

14 Supplemental methods: qpcr analysis 1. Standard Curves relative expression values are calculated Dilution series MIR163 Set1 Set 2 log conc TechRep1 TechRep2 TechRep3 mean Ct TechRep1 TechRep2 TechRep3 mean Ct mean Ct both sets MIR172a Set1 Set 2 log conc TechRep1 TechRep2 TechRep3 mean Ct TechRep1 TechRep2 TechRep3 mean Ct mean Ct both sets log concentration y = x R² = Ct log concentration a y = x R² = Ct