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1 doi: /nture09470 prmt5-1 prmt5-2 Premture Stop Codon () GGA TGA PRMT5 Hypocotyl Length (Reltive to Drk) ** 30 *** c prmt5-1 d ** *** 150 prmt5-2 ** *** prmt5-1 prmt prmt5-1 prmt5-2 Lef Angle (degrees) Period (h) Supplementry Fig. 1. Structure of PRMT5 nd physiologicl chrcteriztion of prmt5 mutnts. () Structure of the PRMT5 gene nd scheme of the PRMT5 protein. Introns re represented y lines nd exons y oxes (gry oxes indictes coding regions nd lck oxes, UTR s). T-DNA insertions in prmt5-1 nd prmt5-2 mutnts nd nonsense muttion in re indicted. The premture stop codon generted in mutnt spns exon 20 s lst se, in red, nd the two ses t the eginning of exon 21, in lck (Upper Pnel). Digrm of the PRMT5 protein. Functionl nd conserved PRMT5 domin is in lue. The frgment sent in the protein from mutnt is represented y crossed lines (Lower Pnel). () Hypocotyl length of wild-type nd prmt5 seedlings grown under short dys (SD, 8h light: 16h drk) reltive to hypocotyl length of the sme genotypes under constnt drk conditions (n=6 replictes of 10 seedlings ech). (c) Circdin rhythm of lef movement under constnt light (LL; h, hours) (n=6)., wild-type (Columi ecotype);, prmt5-1 mutnt nd, prmt5-2 mutnt. (d) Period length of the circdin rhythm of lef movement estimted for the indicted genotypes y using FFT-NLLS (h, hours; n=6). Error rs indicte +SEM (*** P<01, ** P<1). 1

2 Rel-Amp TOC1::LUC Period (h) TOC1 (PRR1) /ACT Time in Long Dys (h) TOC1 (PRR1) /ACT c Supplementry Fig. 2. Circdin oscilltions in TOC1 expression in prmt5 mutnts., Period length nd reltive mplitude error estimted for TOC1::LUC ioluminescence rhythms y using FFT-NLLS nlysis. Seedlings were entrined under 12h light: 12h drk cycles for 7 dys efore eing relesed into constnt light conditions for 5 dys. TOC1 expression mesured y qpcr in seedlings kept under long dy (16h light:8h drkness) conditions, n=5 () or under LL fter entrinment under 12h light: 12h drkness (c); vlues re expressed reltive to ACTIN8 nd normlized to the mximum vlue of ech gene. Dt represent verge +SEM., wild-type (Columi ecotype) nd, mutnt. 2

3 LHY/ACT Time in Long Dys (h) LHY/ACT CCA1/ACT c Time in Long Dys (h) CCA1/ACT d Supplementry Fig. 3. PRMT5 ffects dily nd circdin oscilltions of CCA nd LHY expression.,, LHY nd c, d, CCA1 expression mesured y qpcr in seedlings kept under long dy (16h light:8h drkness) conditions, n=5 (, c) or under LL fter entrinment under 12h light: 12h drkness,n=4 (, d); vlues re expressed reltive to ACTIN8 nd normlized to the mximum vlue of ech gene. Dt represent verge +SEM., wild-type (Columi ecotype) nd, mutnt. 3

4 PRMT5/ACT 1.4 : Col : Ler c Time in SD (h) PRMT5 (Expression Level) Time in SD (h) PRMT5 (Expression Level) : Col Supplementry Fig. 4. Dily nd circdin regultion of PRMT5 expression. () PRMT5 expression mesured y qpcr under short dys (SD; 8h light: 16h drkness). Vlues (n=4) re reltive to ACTIN8 nd represent verge +SEM., wild-type (Columi ecotype). () PRMT5 expression levels determined y microrry nlysis in plnts grown under SD during two consecutive dys. Dt were normlized to the mximum vlue nd were otined from Michel, T.P. et l. PLoS Genet. 4, e14 (2008)., wild-type (Lndserg erect ecotype). (c) Normlized PRMT5 expression levels determined y microrry nlysis in plnts grown under LL fter entrinment under 12h light: 12h drkness. Dt were otined from Edwrds, K.D. et l. Plnt Cell 18, (2006)., wild-type. Open nd closed oxes indicte light nd drk period respectively. Lined oxes indicte sujective night. 4

5 Rosette lef numer *** *** *** flc flc; ** flc *** flc; Period (h) Supplementry Fig. 5. FLC is not responsile for the longer circdin period of the mutnt. Flowering time mesured s the numer of rosette leves t time of 1 cm high flower olt, n=22-24 (left pnel). ANOVA followed y Tukey's Multiple Comprison Test ws used to check for sttisticl significnce in comprisons mong genotypes. Free-running period of lef movement under LL, n=6. h, hours. (right pnel). ***P<01, **P<1. Dt represent verge +SEM. Period estimtes were clculted with Brss 3.0 softwre (Biologicl Rhythms Anlysis Softwre System); ville from ( nd nlyzed with the FFT- NLLS suite of progrms, s descried previously (see Methods). ANOVA test followed y Tukey's Multiple Comprison Test were pplied for comprisons. 5

6 Enrichment Up-regulted in Phse (h) Enrichment Down-regulted in Phse (h) Z score c Up-regulted in Phse (h) Z score d Down-regulted in Phse (h) Supplementry Fig. 6. Phse distriution of clock-regulted genes whose expression ws ffected in mutnt plnts. Phse enrichment of circdin-regulted genes whose expression ws up () or down-regulted () in mutnts. Phse enrichment Z- score of genes up (c) or down-regulted (d) in mutnts (h, hours). The nlysis ws performed with the genes identified s up or down regulted t lest 1,5 fold in prmt5 mutnt plnts (S. tle I), which were lso identified s clock regulted y Edwrds et l. Plnt Cell, 18, 639 (2006). The phse over-representtion nlysis ws conducted with Phser ( nd ws sed on phses of expression estimted using the LL23 (i.e. 2 nd nd 3 rd dys under free-running conditions) dtset (Michel et l. Plos Biology, 6, e225 (2008). 6

7 PRR9 (Expression Level) W T prm t5-5 PRR9/ACT Supplementry Fig. 7. PRMT5 ffects expression of the clock gene PRR9., PRR9 expression mesured y Affymetrix microrrys in plnts grown lwys under LL (n=3). Dt represents verge +SEM. White r, wild-type; lck r, mutnt., PRR9 expression mesured y qpcr, in seedlings trnsferred to LL fter entrinment under light:drk cycles (n=4). Dt represents verge +SEM., wild-type;, mutnt. Open nd lined oxes indicte sujective dy or night period respectively. 7

8 ZT (h) FLC PRR9 Supplementry Fig. 8. Symmetric dimethyltion of histone 4 y PRMT5. Symmetric dimethyltion of histone 4 rginines in the regultory regions of FLC nd PRR9 ws evluted y Re-ChIP ssys. Chromtin ws immunoprecipitted using nti histone H4 followed y immunoprecipittion with nti-symmetric dimethyl-rginine ntiodies. Representtive PCR nds from Re-ChIP ssys in seedlings entrined under 12h light:12h drkness re shown t two different times of the light/drk cycle. (-) without DNA control, (+) input DNA control. 8

9 Supplementry Fig. 9. PRMT5 is required for symmetric rginine dimethyltion of Sm proteins in Aridopsis., Western lot nlysis of lystes from nd mutnt seedlings grown under light/drk cycles, proed with the symmetric dimethyl rginine-specific ntiody SYM10. This ntiody detects symmetriclly dimethylted rginines nd recognized three smll proteins in smples from ut not, or to lesser extent, in smples from prmt5 mutnt plnts. Arrows show nds nerly sent, or significntly reduced, in the mutnt. Bsed on similr findings with SYM10 in flies, these smll proteins re likely Sm nd/or LSm proteins., Comprison of SYM10 rectivity in smples from nd SmD3 overexpressing lines (35S:SmD3). The rrow indictes the loction of SmD3. The higher rectivity otined with SYM10 ntiodies in the SmD3 over-expressing lines indictes tht SmD3 is indeed trget of PRMT5 ctivity in Aridopsis. SmD3 levels in the plnts used for these experiments were close to the limit of detection of the SYM10 ntiody. Left numers represent moleculr weights (KD). 9

10 Supplementry Fig. 10. Alterntive splicing of the clock gene PRR9 is ffected in different prmt5 mutnt lleles. Alterntive PRR9 mrna vrints with intron 3 retined or spliced were mesured y rdioctive PCR in wild-type, nd prmt5-1 mutnt plnts grown under LL without ny prior temperture or light entrinment. A representtive slice of gel is shown. 10

11 Period (h) PRR9_B OX PRR9 (Reltive Units) Supplementry Fig. 11. Overexpression of PRR9_B does not ffect circdin rhythms in lef movement in Aridopsis plnts. Period of circdin rhythm of lef movement under constnt light nd temperture conditions in wild-type ( ) or PRR9_B OX ( ) seedlings. PRR9_B over-expressing seedlings re independent T1 lines of trnsgenic plnts crring construct tht overexpress the truncted isoform of PRR9 originted y the premture stop codon present in oth PRR9_B nd PRR9_D isoforms (h, hours; n=5). Expression level of PRR9 ws mesured y qpcr nd represents oth, endogenous nd exogenous PRR9 isoforms. Vlues re expressed reltive to PP2A. Dt is representtive of three independent experiments. Dotted line indictes men period vlue of plnts. 11

12 PRR9_A/ACT PRR9_B/ACT PRR9_A/PRR9_B c Supplementry Fig. 12. PRMT5 ffects the lterntive splicing of the clock gene PRR9. Expression of PRR9_A () nd PRR9_B () y qpcr under LL (n=4). Vlues re reltive to ACTIN8. c, A/B isoforms rtio s mesure of PRR9 s lterntive splicing pttern. Dt represents verge +SEM., wild-type;, prmt5 mutnt. 12

13 PRR7/ACT Time in Long Dys (h) PRR7/ACT Supplementry Fig. 13. PRMT5 ffects dily nd circdin oscilltions of PRR7 expression. qpcr mesurements of PRR7 expression in seedlings kept under long dy (16h light:8 h drkness) conditions, n=5 () or under LL fter entrinment under 12h light: 12h drkness, n=4 (); vlues re expressed reltive to ACTIN8 nd normlized to the mximum vlue of ech gene. Dt represent verge +SEM., wild-type (Columi ecotype) nd, mutnt. 13

14 PCR with intron-flnking primers Supplementry Fig. 14. Vlidtion y conventionl RT-PCR of intron retention events identified using tiling rrys in Aridopsis. gdna, genomic DNA (control); +RT indictes rections with reverse trnscriptse (RT). As control, rections without RT (-RT) were performed. The upper nd corresponds to the unspliced isoform (s in gdna) nd the lower nd to the spliced one. Dt is representtive of three independent experiments. 14

15 67% 23% At2g SS/Intron Retention 252 p X 332 p 339 p 244 p 7% 3% 57% 10% 17% 16% Supplementry Fig. 15. Electropherogrm chrcterizing splicing pttern chnges of PRR9 (At2g46790) in wild-type nd mutnt plnts. Schemtic gene structures showing the expected lterntive spliced isoforms nd their corresponding sizes. Percentge of ech isoform reltive to the totl is shown. 15

16 RSp31 mrna3/mrna RSp31 mrna3/mrna RSp31 mrna3/mrna c prr7;prr RSp31 mrna3/mrna d prr7;prr9,prmt RSp31 mrna3/mrna1 e At5g57630 Distl 5'/Totl f Time in SD (h) Supplementry Fig. 16. Alterntive splicing of RSp31 nd At5g57630 is ffected in prmt5 mutnts. -e, RSp31 splice vrints mesured y rdioctive RT-PCR in seedlings trnsferred to LL fter entrinment under light:drk cycles (-d) or in plnts grown under SD (8h light: 16h drkness) (e). Different isoforms of this gene hve een previously reported (Klyn, M. et l. Nucl. Acids Res. 34, (2006)). At5g57630 splice vrints mesured y rdioctive RT-PCR in seedlings trnsferred to LL fter entrinment under light:drk cycles (f). Vlues re verge +SEM (n=3)., wild-type;,., prr7;prr9;, prr7;prr9;prmt5. Open nd closed oxes indicte light nd drk period respectively. Lined oxes indicte sujective night. 16

17 csul RM 50 /+ RM 50 csul Rhythmicity (%) ** Averge DD FFT * 0 csulrm50/+ csulrm50 0 csulrm50/+ csulrm50 Supplementry Fig. 17. PRMT5 regultes circdin rhythms in locomotor ctivity in Drosophil. () Representtive doule-plotted ctogrms for the indicted genotypes. Trnsfer to constnt conditions (DD) ws performed fter four dys. The lck nd white oxes t the top of the ctogrms indicte night nd dy phses, respectively. The lck nd gry rs t the ottom of the ctogrms indicte sujective night nd dy respectively. () Percentge of rhythmic flies (left) nd power FFT (right), mesure of the strength of the rhythm, in the different genotypes. Error rs indicte SEM. Error rs indicte +SEM (* P<5, ** P<1; n=53-73). 17

18 clk/rp drt Time in DD (h) tim/rp drt Time in DD (h) Pdp1/rp49 c 1.4 drt Time in DD (h) Supplementry Fig. 18. Circdin regultion of core-clock gene expression in drt5 mutnt flies. clk (), tim () nd Pdp1 (c) expression in fly-heds mesured y qpcr. Flies were entrined under 12h light:12h drkness cycles nd then relesed into DD (h, hours). Vlues re expressed reltive to rp49 nd normlized to the mximum of ech time-course nd ech genotype. Dt re verge +SEM of 4 replictes., wild-type nd, drt5 mutnt. 18

19 to Retined/Spliced (fold effect) yw per 01 CT2 CT14 jrk CG14180 Retined/Spliced (fold effect) yw per 01 CT2 CT14 jrk Supplementry Fig. 19. PRMT5-regulted lterntive splicing events re not ffected in rrhythmic clock mutnts. to () nd CG14180 () splice vrints in fly-heds mesured y rdioctive RT-PCR in DD; dt re verge +SEM, n=2 (representtive of two independent experiments). CG7199, gene whose lterntive splicing ws lso ffected in drt5 (Fig. 4f) ws lso nlyzed in rrhythmic clock mutnts ut ws excluded from this nlysis due to its low expression in the yw ckground. 19

20 to (Expression level) to/rp drt5 0 drt Time in DD (h) Supplementry Fig. 20. Expression of tkeout, clock-output gene, is ltered in drt5 mutnt flies. tkeout (to) expression mesured using ffymetrix rrys, n=3 () or y qpcr in DD, n=4 (). Vlues re expressed reltive to rp49. The lck nd gry rs t the ottom indicte sujective night nd dy respectively., wild-type (w 1118 );, drt5. Dt re verge +SEM. 20

21 Supplementry Fig. 21. Overlp etween genes under PRMT5 nd circdin clock control in DD. Numer of genes whose expression ws ltered in drt5 nd re under circdin clock control in constnt drkness (Dt were otined from Keegn, K.P. et l. PLoS Comput Biol 3, e208 (2007). The overlp etween these groups is shown. The representtion fctor (RF) is the oserved/expected overlp, nd p ws clculted using the hypergeometric test. 21

22 drt5/rp Time in DD (h) Supplementry Fig. 22. drt5 expression is not regulted y the circdin clock under constnt drkness. drt5 expression mesured y qpcr in cdna smples otined from flyheds. Flies were entrined in 12h light:12h drkness cycles nd then relesed to DD (h, hours). Vlues re expressed reltive to rp49 nd normlized to the mximum vlue. Dt re verge +SEM of 4 replictes., wild-type (w 1118 ). 22

23 Supplementry Fig. 23. Overlp etween PRMT5 nd CLOCK regulted genes. Numer of genes controlled y drt5 nd Clk (jrk), nd the overlp etween those up-regulted in drt5 nd down-regulted in Clk () nd the other wy round (). 23