Supplementary Information

Transcription

1 Supplementary Information COP1 E3 ligase protects HYL1 to retain microrna biogenesis Seok Keun Cho 1, Samir Ben Chaabane 1, Pratik Shah 1, Christian Peter Poulsen 1, and Seong Wook Yang 1 * 1 Laboratory of Plant Biochemistry, Department of Plant and Environmental Sciences, Center for UNIK Synthetic Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark, Equal contribution * Correspondence to S.W.Y. (swyang@life.ku.dk)

2 Emission Intensity (A.U.) Emission Intensity (A.U.) Emission Intensity (A.U.) Emission Intensity (A.U.) Emission Intensity (A.U.) a c 3.5x x x x x x x x x x x x x x x x x x mir166 sensor Wavelength (nm) b d 3.0x mir172 sensor 2.5x x x x x Wavelength (nm) 5.5x x10 6 Col-0 total RNAs x x hyl1-2 total RNAs 340 cop1-6 total RNAs x x x x x x x x x x x x x x x x x x Wavelength (nm) Wavelength (nm) Wavelength (nm) e Supplementary Figure 1 Photoluminescence properties of two DNA/AgNCs probes and total RNAs (a) Emission spectra of mir166 probe (15 ) in Tris-acetate buffer (20 mm), 1 hour after AgNCs creation, as a function of excitation wavelength (from 300 to 720 nm, in 20 nm step size). (b) Emission spectra of mir172 probe (15. The sensors show their unique emission profiles. These results show the unique fluorescence properties of two DNA/AgNCs probes. (c) Emission spectra of Col-0 total RNA (5 g. (d) Emission spectra of hyl1-2 total RNA (5 g. (e) Emission spectra of cop1-6 total RNA (5 g. Total RNA from plants are unable to form the highly emissive red and far-red fluorescence, confirming the probes-specific fluorescence. Silver nanoclusters-generated emissions are observed between 0 nm and 7 nm.

3 mirna accumulation by length distributions Read counts a b mirna length distributions cop cop cop cop hyl hyl1-2 Down-regulated mirnas Up-regulated mirnas Length (nt) bc Log 2 (hyl1-2/col-0) Log 2 (cop1-4/col-0) Log 2 (cop1-6/col-0) Supplementary Figure 2 The observed length distribution of mirnas in wild type and mutants (a) Comparison of up-or-down regulated mirnas amongst mutant backgrounds. (b) The x-axis represents various lengths of mirna sequences and the reading frequencies are displayed on y-axis. (c) Different plot of the mirna distributions in mutants.

4

5 Supplementary Figure 3 Morphological and molecular phenotypes of cop1 alleles (a) Phenotype of four week old cop1-4 and wild type plants. (b) Dark grown cop1 alleles show deetiolated phenotypes, open cotyledon and attenuated hypocotyls. (c) Left panel; the levels of LONG HYPOCOTYL IN FAR-RED 1 (HFR1) in cop1 alleles and hfr1-201 mutant. Seedlings were grown under continuous darkness and exposed to far-red light for 1 hour. Right panel; the levels of phytochrome A (phya) in cop1 alleles and wild type. Seedlings were grown for 7 days under a 12-hours white light/12-hours darkness photoperiod and sampled 3 hours after transitioning to white light. These results ensure the deficiency of COP1 E3 ligase activity in cop1 alleles which are used in this study. (d) Variations in the level of HYL1 in individual cop1-6 seedling, a weak cop1 allele. The levels of HYL1 were correlated with the levels of COP1 in four randomly selected 10 day-old cop1-6 seedlings. The levels of endogenous HYL1 and COP1 were determined with and -COP1 antibody, respectively. The equal loading of samples was determined with -actin antibody.

6 WT/35S-HYL1-6myc WT/35S-HYL1-6myc WT/35S-HYL1-6myc a CHX (0.5 mm) Mock PIs (x) MG132( M) (h) b CHX (0.5 mm) Mock CLL PYR (h) ( M) ** * ** * -ACTIN -ACTIN c CHX (0.5 mm) Mock EPX (0.5 M) MG132 (10 M) (h) ** * -ACTIN Supplementary Figure 4 CHX-chase assay of HYL1 using WT/35S-HYL1-6myc transgenic seedlings (a) Effects of PIs or MG132 on HYL1 destabilization. (b) Effects of CLL or PYR-41 on HYL1 destabilization. (c) Effect of epoxomicin on HYL1 destabilization. The levels of endogenous HYL1 and HYL1-6myc were determined with antibody. Single asterisk means endogenous HYL1; double asterisk means HYL1-6myc. In all tests, the equal loading of samples was determined with -ACTIN antibody.

7 Supplementary Figure 5 Effect of chemicals on the formation of acidic vacuolar organelles (AVOs) in wild-type seedlings LysoTracker staining of chemical treated seedlings and confocal analyses; (a) Seedlings were incubated with 3-MA (2 mm). (b) Seedlings were incubated with BTH ( M). (c) Control seedlings without inhibitors. (d) Seedlings were incubated with MG132 ( M). (e) Seedlings were incubated with CLL ( M). (f) Control seedlings without dye. (g) Seedlings were incubated with PIs (2-1 X). All seedlings were stained with LysoTracker for 3 h after incubating 18 h with the indicated chemicals.

8 Relative amount of HYL1 (A.U.) Col-0 Col-0 a CLL (18 h) b Epoxomicin (18 h) ( M) ( M) -ACTIN M M Supplementary Figure 6 HYL1 is not degraded by UPS-coupled autophagy Protein blot analyses of chemically treated seedlings; (a) Seedlings were incubated with CLL (0 - M). (b) Seedlings were incubated with epoxomicin (0-5 M). The levels of HYL1 were determined with antibody. Equal loading of samples was determined with -actin antibody. The relative amounts of HYL1 were calculated by image analysis and plotted in arbitrary unit versus the level of initial sample. The data were collected as average value of four immuno-blot analyses with ±standard deviation (n=4).

9 Cleaved peptides? Col-0 se-1 hyl1-2 cop1-4 cop1-6 HIS-HYL1 MG132 (10 M) a cop1-4 Crude extract PIs MG132 CLL Epoxomicin (h) (h) (h) * HIS b * HYL ox ** * Image of Fig. 2a Intensified image of Fig. 2a Supplementary Figure 7 In vitro cell-free degradation assay using the crude extract of cop1-4 (a) Rapid degradation of recombinant HIS-HYL1 by cop1-4 crude extract and the concentration-dependent effects of chemicals. (b) Uncropped immuno-blot image reveals the cleaved fragments-like products of HYL1 in vivo. Single red asterisk means endogenous HYL1; double red asterisk means HYL1-6myc. Single blue asterisk indicates the traceable amount of HYL1 in cop1-4 in contrast to the total absence in hyl1-2. The levels of HYL1 were determined with antibody.

10 3-MA BTH PIs (E64D) MG132 CLL Epoxomicin PYR-41 Autopha gopore Autopha gosome Serine and cysteine Proteases 26S proteasome Inhibition Autophago lysosome Crosstalk UPS Autophagy Supplementary Figure 8 The effects of chemicals on autophagy and UPS Blue arrow means activation. Red line means inhibition. Three specific UPS inhibitors were ineffective to block HYL1 degradation in vitro and in vivo. PIs and MG132 block the degradation in vitro and in vivo, implying that HYL1 is a target of serine and/or cysteine proteases. BTH, PIs or MG132 increased AVOs (autophagy formation) but highly accumulated HYL1. On the contrary, 3-MA reduced the formation of AVO and HYL1. Based on these results, we suggested that autophagy flux is not responsible for the degradation of HYL1. Further, the presence of a non-autophagic protease, protease X, could be predicted with the results of in vitro cell-free degradation assay.

11 WT/35S-HYL1-CFP/ 35S-At2g303-YFP #5 WT/35S-HYL1-CFP/ 35S-At2g303-YFP #5 Light Light Light HYL1-CFP At2g303 -YFP HYL1-CFP At2g303 -YFP HYL1-CFP At2g303 -YFP Dark Dark Dark HYL1-CFP At2g303 -YFP HYL1-CFP At2g303 -YFP HYL1-CFP At2g303 -YFP Supplementary Figure 9 Visualization of the dark-triggered reduction of HYL1 using WT/35S-HYL1-CFP/35S-At2g303-YFP transgenic seedlings The decaying of HYL1-CFP was monitored in transgenic line #5. Upper panels; CFP and YFP fluorescence under light, Lower panels; CFP and YFP fluorescence after 24 h transferring to darkness. At2g303-YFP was used as an internal control.

12 WT/35S-HYL1-CFP/ 35S-At2g303-YFP Col-0 Col-0 a Light/Dark/Light transition b Col-0 cop1-6 L D L D (h) -ACTIN L D L D L D (h) -Actin c Light/Dark transition 75 L D L D 24(h) ** * #2 #5 -ACTIN Supplementary Figure 10 Light is essential for the stabilization of HYL1 (a) The light/dark/light transition assay. 10 day old wild-type seedlings grown under continuous light (100 μmol m -2 s -1 ) were transferred to continuous dark for a day and then move back to the continuous light for a day to stimulate the in-and-out remobilization of COP1. (b) The light/dark/light transition assay 10 day old cop1-6 seedlings grown under continuous light (100 μmol m -2 s -1 ) were transferred to continuous dark for a day and then move back to the continuous light for a day. The level of HYL1 was determined with antibody. (c) Immuno-blot analysis of the transgenic HYL1-CFP. Double asterisk means HYL1-CFP; single asterisk means endogenous HYL1. In all tests, the equal loading of samples was determined with -ACTIN antibody.

13 Col-0 a PIs (18h) MG132 (18h) Col-0 cop1-6 Col-0 cop (x) ( M) b MG132 (18h) cop ( M) -ACTIN -ACTIN Supplementary Figure 11 Effects of PIs or MG132 on the stability of HYL1 in cop1-6 (a) Indicated concentration of PIs and MG132 were treated to 14 day grown cop1-6 seedlings. (b) The treatment of MG132 was highly effective on cop1-6 protoplasts. The level of HYL1 was determined with antibody and the equal loading of samples was determined with - ACTIN antibody.

14 HYL ox HYL ox Col-0 se-1 hyl1-2 cop1-4 cop1-6 Col-0 se-1 hyl1-2 cop1-4 cop SE -SE Supplementary Figure 12 Uncropped immuno-blot image of SERRATE in vivo.

15 Supplementary Dataset 1 Comparison of mirna expression levels among wild type, hyl1-2, cop1-4, and cop1-6 by small RNA sequencing

16 Supplementary Table 1 List of probes, DNA sensors, and primers used in this study DNA probe mir GTGCTCACTCTCTTCTGTCA-3 mir GTGCTCTCTATCTTCTGTCAA-3 mir TGCTTTGTCTACATTTGGGA-3 mir TAGAGCTCCCTTCAATCCAAA-3 mir TGGCATACAGGGAGCCAGGCA-3 mir TGCACGTGCCCTGCTTCTCCA-3 mir GGGGAATGAAGCCTGGTCCGA-3 mir TTCCCGACCTGCACCAAGCGA-3 mir CAGGCAAGTCATCCTTGGCTA-3 mir GATATTGGCGCGGCTCAATCA-3 mir ATGCAGCATCATCAAGATTCT-3 mir GTGATTTCTCTCTGCAAGCGAA-3 U6snRNP 5 -GCTAATCTTCTCTGTATCGTTCC-3 DNA sensor mir166 sensor 5 -CCTCCTTCCTCCGGGGAATGAAGCCTGGTCCGT-3 mir172 sensor 5 -GGATGCAGCATCATCAAGATTCTCCTCCTTCCTCC-3 QRT-PCR ACTIN2 (At3g18780) 5 -GCACCCTGTTCTTCTTACCG-3 /5 -AACCCTCGTAGATTGGCACA-3 SERRATE (At2g27100) 5 -AAGGCATGGTATGGGTGGAG-3 /5 -AAGGGCTTGTGTTTGTTCAACG-3 HYL1 (At1g09700) 5 -CACCATGACCTCCACTGATGTT-3 /5 -CTCACTGGATTTTGCTAATTCC-3 RT-PCR SERRATE (At2g27100, 741 bp) 5 -CACCATGGCCGATGTTAATCTTCCTCCG-3 /5 -ATACTTATTTTTCAACCAGTCTTCCTC-3 HYL1 (At1g09700, 1260 bp) 5 -CACCATGACCTCCACTGATGTT-3 /5 -TTATGCGTGGCTTGCTTCTGTC-3 DCL1 (AT1G01040, 780 bp) 5 -GTCACCACGCTGTCAAGAAACATCC-3 /5 -CTGCGGCGCCTATTACTTCATCAAC-3 UBC10 (AT5G53300, 466 bp) 5 -TGGATATGGCGTCGAAGC-3 /5 -GTGGGATTTTCCATTTAGCC-3 TUB8 (AT5G23860, 207 bp) 5 -ATAACCGTTTCAAATTCTCTCTCC-3 /5 -TGCAAATCGTTCTCTCCTTG-3

17 Construction COP1 F COP1 R COP1 283R COP1 375F 5 -CACCATGGAAGAGATTTCGACGGATC-3 5 -CGCAGCGAGTACCAGAACTT-3 5 -CGAATCTGACCCACTCAGCG-3 5 -CACCATGTCAAGCATAGAGTTTGATCGTGA-3

18 Supplementary Table 2 Statistical analysis of decaying of HYL1-CFP in darkness Light Dark Mean 1.24 Mean 0.48 SD 0.84 SD 0.35 Cells 75 Cells 161 P value and statistical significance: the two-tailed P value is less than By conventional criteria, this difference is considered to be extremely statistically significant. Confidence interval: the mean of Light minus Dark equals , 95% confidence interval of this difference: From to Intermediate values used in calculations: t = , df = 238, standard error of difference = Light: Dark: Pixel by pixel ROIs Count 3078 Count 79 Mean 1.15 Mean 1.24 SD 0.7 SD 0.84 Pixel by pixel ROIs Count 6664 Count 161 Mean 0.41 Mean 0.48 SD 0.31 SD 0.35