SUPPLEMENTARY INFORMATION

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doi:.8/nture978 Tip6 k RT- w 8 Clk Jrk per tyf e tyf Δ tyf/cg87 GlcAT-I tyf-n tyf G87 tyf ΔG + + ~. k deletion tyf-c Tip6 tyf e6 + GlcAT-I tyf-n tyf-c Supplementry Figure.

1 doi:.8/nture978 Tip6 k RT- w 8 Clk Jrk per tyf e tyf Δ tyf/cg87 GlcAT-I tyf-n tyf G87 tyf ΔG + + ~. k deletion tyf-c Tip6 tyf e6 + GlcAT-I tyf-n tyf-c Supplementry Figure. Moleculr chrcteriztion of tyf mutnts., Schemtic digrm of tyf mutnt lleles. Drk gry oxes, exons; light gry oxes, untrnslted regions; sterisk, stop codon generted y deletion in tyf llele., Semi-quntittive trnscript nlyses in wild-type, clock nd tyf mutnt fly heds. AlldChoe Supplementry Figure

doi:.8/nture978 TYF 9 79 67 Supplementry Figure. tyf is conserved in Drosophil species nd other insects., Schemtic digrm of TYF protein.

2 doi:.8/nture978 TYF Supplementry Figure. tyf is conserved in Drosophil species nd other insects., Schemtic digrm of TYF protein. Highly conserved domins in Drosophil tyf homologs re indicted y lck oxes. A multiple lignment of one of conserved domins is shown elow., Phylogenetic nlysis of tyf homologs. Amino cid sequences of tyf homolog were used to uild the phylogenetic tree with AroDrw softwre ( AlldChoe Supplementry Figure

3 doi:.8/nture978 c tyf-c Tip6 GlcAT-I +/+ tyf e /+ RTtyf e /tyf e Df()HC/+ Df()HC/tyf e Df()r/+ Df()r/tyf e Power of rhythmicity (P-S) 8 6 cyc +/+ tyf Δ /+ tyf e /+ tyf Δ /tyf Δ tyf e /tyf e tyf Δ /tyf e Df()HC/+ tyf Δ /Df()HC tyf e /Df()HC Df()r/+ tyf Δ /Df()r tyf e /Df()r tyf e /+ (n=9) 97% Rhythmic. +/-. h Df()r/tyf e (n=) 68% Rhythmic 9. +/-. h Df()HC/tyf e (n=9) 9% Rhythmic 6.9 +/-. h Supplementry Figure. Deficiency lines fil to complement tyf hypomorphic lleles., Semiquntittive trnscript nlyses in trns-heterozygotic tyf mutnt flies. Totl RNA ws prepred from femle fly heds nd used in RT-PCR with gene-specific primers indicted t the left. Genotypes re shown t the top. RT-, reverse trnscriptse-negtive control., Averged ctogrm nlyses during LD nd 7DD cycles. Genotypes, numers of flies tested, percentge of rhythmic flies, nd period +/- stndrd errors of the men (SEM) under free-running conditions re given t the left. White/lck rs, LD cycle; Gry/lck rs, DD cycle. c, Complementtion ssys in trns-heterozygotic tyf mutnt flies. tyf hypomorphic flies were crossed with wild-type, tyf mutnt, or deficiency lines nd their virgin progeny ws tested in the ehviourl nlyses. P-S vlues, mesure of rhythmic strength under constnt drkness condition, were verged from individul flies with given genotype. Ech fly with P-S vlues greter thn (red dotted line) is defined s rhythmic. Error rs indicte SEM. AlldChoe Supplementry Figure

g tyf>gfp LNd l-lnv s-lnv e tyf>gfp PER tyf/cyc..8.

. w8 ZT tyfe ZT6 tyf per ZT ClkJrk ZT8 DN DN

6 d tyf>gfp PER h PER tyf/cyc trnscript level

-f, The sptil expression of tyfgal trnsgene in

expression (green), while clock cells re

(posterior rin) re enlrged on the lower pnels -d

LNd, dorsl lterl neuron; l-lnv, lrge ventrl lterl

were mesured from heds RNA t the indicted

using reltime RT-PCR nd normlized to non-cycling

Reltive trnscript level t ech ZT ws clculted y

performed using heds RNA of wild-type, tyf, nd

4 g tyf>gfp LNd l-lnv s-lnv e tyf>gfp PER tyf/cyc ZT ZT7 ZT ZT ZT ZT ZT9 ZT.. w8 ZT tyfe ZT6 tyf per ZT ClkJrk ZT8 DN DN tyf>gfp PER Merge PER f Clk/cyc. TYF-V i DN.6 d tyf>gfp PER h PER tyf/cyc trnscript level tyf>gfp c Reltive trnscript level doi:.8/nture978 Supplementry Figure. Sptil nd temporl expression of tyf. -f, The sptil expression of tyfgal trnsgene in dult fly rins is visulized y GFP reporter expression (green), while clock cells re identified y immunostining with nti-per ntiody (red). White oxes on the pnels (nterior rin) nd e (posterior rin) re enlrged on the lower pnels -d nd f to show ech group of clock cells. LNd, dorsl lterl neuron; l-lnv, lrge ventrl lterl neuron; s-lnv, smll ventrl lterl neuron; DN, dorsl neuron. g, Clk (oxes) nd tyf (tringles) trnscript levels were mesured from heds RNA t the indicted Zeitgeer times (ZT, lights-on; ZT, lights-off) using reltime RT-PCR nd normlized to non-cycling cyc trnscript level. Reltive trnscript level t ech ZT ws clculted y normlizing to wild-type level t ZT set to. Error rs indicte SEM. h, Quntittive trnscript nlyses were similrly performed using heds RNA of wild-type, tyf, nd clock mutnt flies t ZT (white rs) nd ZT (lck rs) under LD cycle. i, C-terminlly V-tgged TYF ws expressed in PDF+ cells of tyfe mutnt fly. Adult rins were dissected t the indicted ZTs nd immunostined with nti-v (green) nd nti-per (red) ntiodies. Imges show the signls from smll LNvs. AlldChoe Supplementry Figure

doi:.8/nture978 c ETOH RU86 w 8 tyf e tyf DP POT 6 6 6 w 8 ;;ElvGS>TYF tyf ;;ElvGS>TYF tyf e ;;ElvGS>TYF PDF intensity (%) 8 6 w 8 tyf tyf e ZT

Pcemker neurons re normlly developed in tyf hypomorphic flies., PDF projections from ventrl lterl neurons (LN v s).

DP, dorsl projection from smll LN v s; POT, posterior optic trct from lrge LN v s.

c, Adult-specific TYF overexpression rescues ehviourl rhythms in tyf mutnt flies.

Locomotor ctivity ws monitored during susequent LD nd 7DD cycles.

5 doi:.8/nture978 c ETOH RU86 w 8 tyf e tyf DP POT w 8 ;;ElvGS>TYF tyf ;;ElvGS>TYF tyf e ;;ElvGS>TYF PDF intensity (%) 8 6 w 8 tyf tyf e ZT ZT Power of rhythmicity (P-S) 8 6 w 8 ;; ElvGS> TYF EtOH tyf ;; ElvGS> TYF RU86 tyf e ;; ElvGS> TYF Supplementry Figure. Pcemker neurons re normlly developed in tyf hypomorphic flies., PDF projections from ventrl lterl neurons (LN v s). Adult rins were dissected t ZT nd stined with nti-pdf ntiody (green). Two representtive imges re shown. DP, dorsl projection from smll LN v s; POT, posterior optic trct from lrge LN v s., PDF intensity in nerve terminls of smll LN v s ws quntified, verged from -6 hemispheres, nd normlized to tht of wild-type t ZT. c, Adult-specific TYF overexpression rescues ehviourl rhythms in tyf mutnt flies. TYF overexpression in dult rin neurons ws driven y Elv-Gene Switch (GS)-GAL driver only fter individul fly ws fed with food contining. mm RU86. Locomotor ctivity ws monitored during susequent LD nd 7DD cycles. As vesicle control, % ethnol ws included in food for control flies with the sme genotypes. Top, verged ctogrm nlyses during LD nd 7DD cycles. White/lck rs, LD cycle; Gry/lck rs, DD cycle. Bottom, verged P-S vlues, mesure of rhythmic strength under constnt drkness condition. Ech fly with P-S vlues greter thn (red dotted line) is defined s rhythmic. Error rs indicte SEM.p<., p<. s determined y Student s t test AlldChoe Supplementry Figure

6 doi:.8/nture978 w 8 tyf Δ tyf e PER intensity (%) PER intensity (%) PER intensity (%) DN DN DN ZT ZT ZT ZT8 ZT ZT6 Supplementry Figure 6. tyf is crucil for PER cycling in pcemker neurons. Adult rins were dissected t the indicted ZTs under LD cycles nd immunostined with nti-per nd nti- PDF ntiodies. The intensity of nti-per stining signls in ech clock cell group ws quntified using ImgeJ softwre, verged from 7- hemispheres t ech ZT, nd normlized to the vlue of wild-type fly t ZT which ws set s %. Error rs indicte SEM. DN, dorsl neuron. AlldChoe Supplementry Figure 6 6

7 doi:.8/nture978 ZT ZT PER intensity (%) DN PER intensity (%) DN PER intensity (%) DN PER intensity (%) 8 6 PDF - Supplementry Figure 7. PER cycling in pcemker neurons requires tyf in cellutonomous mnner. Adult rins were dissected t ZT (white rs) or ZT (lck rs) under LD cycles nd immunostined with nti-per nd nti-pdf ntiodies. The intensity of nti-per stining signls in ech clock cell group ws quntified using ImgeJ softwre, verged from 7- hemispheres t ech ZT, nd normlized to the vlue of wild-type fly t ZT which ws set s %. Error rs indicte SEM., dorsl lterl neuron;, lrge ventrl lterl neuron;, smll ventrl lterl neuron; DN, dorsl neuron. AlldChoe Supplementry Figure 7 7

doi:.8/nture978 TIM CLK CLK PDF TIM intensity (%) w 8 8 6

PDF - DN DN DN per PDP intensity 6 ZT ZT9 w 8 tyf tyf e w

Clock gene expression in tyf mutnt flies.

Adult rins were dissected t ZT nd immunostined with

Asterisks indicte PDF-negtive smll LN v s.

8- hemispheres, nd normlized to tht of wild-type set s %

, dorsl lterl neuron; l-lnv, lrge ventrl lterl neuron;

, CLK protein is comprly expressed in clock cells of

nti-clk (red) nd nti-pdf (green) ntiodies.

oserved in Clk Jrk mutnt fly, were indicted y sterisks.

tyf mutnt flies is comprle to wild-type.

immunostined with nti-pdp nd nti-pdf ntiodies.

using ImgeJ softwre nd verged from -9 hemispheres.

d, tim nd PdpƐ trnscripts normlly cycle in tyf

Lrvl rins were dissected t the indicted time during first

8 doi:.8/nture978 TIM CLK CLK PDF TIM intensity (%) w tyf w 8 tyf tyf e Clk Jrk l-lnv tyf e w 8 tyf tyf e PDF + PDF - DN DN DN per PDP intensity 6 ZT ZT9 w 8 tyf tyf e w 8 tyf tyf e DN DN DN Supplementry Figure 8. Clock gene expression in tyf mutnt flies., TIM expression in wild-type nd tyf mutnt flies. Adult rins were dissected t ZT nd immunostined with nti-tim (red) nd nti-pdf (dt not shown) ntiodies. Asterisks indicte PDF-negtive smll LN v s. TIM intensity in ech cell group ws quntified, verged from 8- hemispheres, nd normlized to tht of wild-type set s % (ottom pnel). Error rs indicte SEM., dorsl lterl neuron; l-lnv, lrge ventrl lterl neuron; s-lnv, smll ventrl lterl neuron. DN, dorsl neuron., CLK protein is comprly expressed in clock cells of wild-type nd tyf mutnt flies. Adult rins were dissected t ZT nd immunostined with nti-clk (red) nd nti-pdf (green) ntiodies. Non- CLK-specific signls from PDF-negtive cells, lso oserved in Clk Jrk mutnt fly, were indicted y sterisks. c, Rhytmic expression of PDP protein in pcemker cells of tyf mutnt flies is comprle to wild-type. Adult rins were dissected t the indicted ZTs nd immunostined with nti-pdp nd nti-pdf ntiodies. Anti- PDP stining intensity in smll LN v s ws quntified using ImgeJ softwre nd verged from -9 hemispheres. Error rs indicte SEM. d, tim nd PdpƐ trnscripts normlly cycle in tyf hypormorphic mutnts. Lrvl rins were dissected t the indicted time during first DD cycle. In situ hyridiztion ssy ws performed using ntisense RNA proes for tim (top) or PdpƐ (ottom) trnscripts. CT, circdin time; DN, dorsl neuron; l-ln, lrvl lterl neuron. d per, tyf e tim PdpƐ CT CT CT CT AlldChoe Supplementry Figure 8 c DN l-ln DN l-ln DN DN DN l-ln l-ln DN l-ln l-ln 8

9 doi:.8/nture978 Pdf-GAL/+ tyf e /Y; Pdf-GAL/+ tyf e /Y; Pdf-GAL/+; UAS-mGFP/+ tyf e /Y; Pdf-GAL/+; UAS-per6/+ tyf e /Y; Pdf-GAL/ UAS-TYF-V tyf /Y; Pdf-GAL/+ tyf /Y; Pdf-GAL/+; UAS-mGFP/+ tyf /Y; Pdf-GAL/+; UAS-per6/+ tyf /Y; Pdf-GAL/ UAS-TYF-V Supplementry Figure 9. PER overexpression rescues ehviourl phenotype in tyf mutnt flies. Averged ctivity profiles during LD cycles (middle pnel) nd verged ctogrms throughout the ehviourl nlyses (right pnel) re shown. Genotypes re given t the left. More detiled ehviorl nlyses in these flies re shown in Tle S. Error rs indicte SEM. While/lck rs, LD cycle; Gry/lck rs, DD cycle. AlldChoe Supplementry Figure 9 9

doi:.8/nture978 GFP GFP PDF per>gfp; Clk Jrk tyf e ; per>gfp tyf ;

per promoter ctivity is not ffected y tyf muttion.

(green) in wild-type, tyf nd Clk Jrk mutnts.

10 doi:.8/nture978 GFP GFP PDF per>gfp; Clk Jrk tyf e ; per>gfp tyf ; per>gfp per>gfp Supplementry Figure. per promoter ctivity is not ffected y tyf muttion. GAL expression driven y per promoter ws monitored y GFP reporter gene (green) in wild-type, tyf nd Clk Jrk mutnts. Lrge LN v s in dult rins were identified y co-stining with nti-pdf ntiody (red). Note tht the per>gfp expression ws diminished in Clk Jrk mutnt, vlidting tht per-gal expression reflects endogenous per promoter ctivity in the clock cells. AlldChoe Supplementry Figure

, PER protein ws expressed from single copy of per

pnel) or per, tyf e doule mutnt (lower pnel).

k genomic region is missing the endogenous trnscription

expressed from n enhncer-trpped heterologous gene

expresses PER( ) protein t constitutively high levels

degrdtion; Pdf>PER, Pdf- GAL drives PER expression from

, PER overexpressed in PDF+ cells of wild-type nd tyf e

11 doi:.8/nture978 perg 7.:9 per(δ)-hahis Pdf>PER Pdf>PER tyf e /Y; Pdf>PER per ZT per,tyf e ZT Supplementry Figure. tyf posttrnscriptionlly regultes per expression., PER protein ws expressed from single copy of per trnsgenes (shown t the top) in per single mutnt (upper pnel) or per, tyf e doule mutnt (lower pnel). Adult rins were immunostined with nti-per (red) nd nti-pdf (dt not shown) ntiodies t ZT. perg, genomic per trnsgene tht expresses wild-type PER protein from. k per locus; 7.:9, promoterless per trnsgene in which 7. k genomic region is missing the endogenous trnscription strt site nd the first noncoding exon, nd PER is expressed from n enhncer-trpped heterologous gene promoter; per( )- HAHis, genomic per trnsgene tht expresses PER( ) protein t constitutively high levels throughout LD cycles due to lck of DBT-medited PER degrdtion; Pdf>PER, Pdf- GAL drives PER expression from UAS-per6 trnsgene in PDF+ cells., PER overexpressed in PDF+ cells of wild-type nd tyf e mutnts were detected y nti-per ntiody t ZT (upper pnel) nd ZT (lower pnel)., dorsl lterl neuron;, lrge ventrl lterl neuron;, smll ventrl lterl neuron; sterisks, PDF-negtive smll LN v s. AlldChoe Supplementry Figure

doi:.8/nture978 TYF TYF-N (-67) TYF-C (8-9) Input GST GST-PABP (kd) 7 7 GST GST-PABP input + GSH pull-down + m7-gtp pull-down + + RNse + eife PABP Ge- TYF-V TYF-xHA Supplementry Figure.

12 doi:.8/nture978 TYF TYF-N (-67) TYF-C (8-9) Input GST GST-PABP (kd) 7 7 GST GST-PABP input + GSH pull-down + m7-gtp pull-down + + RNse + eife PABP Ge- TYF-V TYF-xHA Supplementry Figure. TYF specificlly ssocites with cp-inding complex nd PABP., GST or GST-PABP on ffinity eds ws incuted with S-leled TYF or deletion mutnt protein. Bound proteins were resolved y SDS-PAGE nd detected y utordiogrphy. Coomssie stining of GST proteins is shown on the right. Input, % of S-leled proteins included in ech inding rection., S cell extrcts expressing epitope-tgged TYF were incuted with glutthione sephrose (lne ) or m7-gtp sephrose (lne -) eds. Where indicted,. mg/ml of RNse A ws included in the extrcts nd incuted t room temperture for min prior to the ddition of ffinity eds. Bound proteins were proed with different ntiodies shown on the left. Input, % of extrct included in ech pull-down ssy; GSH, glutthione. AlldChoe Supplementry Figure

S cell extrcts expressing epitope-tgged TYF were frctionted y %-% sucrose density grdient in the sence or presence of mm EDTA.

13 doi:.8/nture978 sucrose grdient % % 8S A S 6S polysomes -EDTA +EDTA TYF-xHA -EDTA +EDTA eife -EDTA +EDTA PABP -EDTA +EDTA -EDTA P +EDTA Supplementry Figure. TYF co-sediments with polyriosomes. S cell extrcts expressing epitope-tgged TYF were frctionted y %-% sucrose density grdient in the sence or presence of mm EDTA. Bsed on the peks in the A profile (top), ech riosoml frction ws identified. Totl protein in ech frction ws further nlyzed y Western lotting with ntiodies shown on the left. AlldChoe Supplementry Figure

doi:.8/nture978 MS MS V TYF-MS TYF-N-MS TYF-C-MS c MS TYF MS TYF-N MS TYF-C MS TYF-MS TYF-N-MS TYF-C-MS 67 9 MS V 6 9 MS V MS V (kd) 6. 97.

... UTR-FLUC-UTR-6xBS MS TYF-MS g 8S rrna levels (%) 8 6 sucrose pellet MS TYF-MS Reltive Fluc RNA levels (pellet/sucrose) UTR-FLUC-UTR-6xBS MS TYF-MS h Activtion fold Ac/TYF-V

, Schemtic digrm of MS nd TYF-MS constructs used in MS RNA-tethering ssys.

Protein size mrkers re shown on the left.

Differentil interference contrst imges re overlid with confocl imges t the ottom pnel. d, per nd tim ' UTR ugment the ctivtion y TYF-MS.

Cells were hrvested nd luciferse ssys were performed t 8 h fter trnsfection.firefly luciferse ctivity (FLUC) ws first normlized to Renill luciferse ctivity (RLUC).

Fluc RNA levels were mesured y reltime RT-PCR nd normlized to Rluc RNA levels. p>.78 s determined y Student s t test.

Fluc RNA levels were first normlized to cyc RNA levels.

g, Trnsfected S cell lystes were frctionted y centrifugtion through % sucrose cushion nd totl RNAs were seprtely purified from sucrose nd pellet frctions.

14 doi:.8/nture978 MS MS V TYF-MS TYF-N-MS TYF-C-MS c MS TYF MS TYF-N MS TYF-C MS TYF-MS TYF-N-MS TYF-C-MS 67 9 MS V 6 9 MS V MS V (kd) V DIC d TYF-dependent ctivtion 8 6 e Fluc RNA levels (%) UTR-FLUC-UTR-6xBS MS TYF-MS f Reltive Fluc RNA levels (cytoplsm/nucleus).... UTR-FLUC-UTR-6xBS MS TYF-MS g 8S rrna levels (%) 8 6 sucrose pellet MS TYF-MS Reltive Fluc RNA levels (pellet/sucrose) UTR-FLUC-UTR-6xBS MS TYF-MS h Activtion fold Ac/TYF-V Ac/TYF-N-V Ac/TYF-C-V UTR-FLUC-UTR-6xBS 6 MS TYF-MS Supplementry Figure. tyf ctivtes gene expression when rtificilly tethered to reporter RNA., Schemtic digrm of MS nd TYF-MS constructs used in MS RNA-tethering ssys., S cell lystes used in luciferse reporter ssys were lso nlyzed y Western lotting with nti-v ntiody to show the expression levels of MS fusion proteins. Protein size mrkers re shown on the left. c, Sucellulr loclztion of MS fusion proteins ws visulized y immunostining with mouse nti-v ntiody nd nti-mouse IgG Alex 9 secondry ntiody (red). Differentil interference contrst imges re overlid with confocl imges t the ottom pnel. d, per nd tim ' UTR ugment the ctivtion y TYF-MS. S cells on -well pltes were cotrnsfected with ng of firefly luciferse reporter contining 'UTR from ech clock gene (Ac/FLUC-UTR-6xBS), ng of Ac/RLUC, nd ng of Ac/MS-V or Ac/TYF-MS-V. Cells were hrvested nd luciferse ssys were performed t 8 h fter trnsfection.firefly luciferse ctivity (FLUC) ws first normlized to Renill luciferse ctivity (RLUC). Activtion fold ws then clculted y normlizing to the vlue in the presence of MS. e, S cells were trnsfected similrly s in Fig. nd totl RNA ws purified t 8 h fter trnsfection. Fluc RNA levels were mesured y reltime RT-PCR nd normlized to Rluc RNA levels. p>.78 s determined y Student s t test. f, Cytoplsmic nd nucler RNAs were seprtely isolted from trnsfected S cells nd trnscript levels were quntified y reltime RT-PCR. Fluc RNA levels were first normlized to cyc RNA levels. The rtio of reltive Fluc RNA levels in cytoplsm frction to those in nucler frction ws compred etween MS nd TYF-MS trnsfected cells. p>.769 determined y Student's t test. g, Trnsfected S cell lystes were frctionted y centrifugtion through % sucrose cushion nd totl RNAs were seprtely purified from sucrose nd pellet frctions. Trnscript levels in ech frction were quntified y reltime RT-PCR. The distriution of 8S rrna in MS or TYF-MS trnsfected cells ws shown on the left pnel, vlidting the comprle frctiontion from MS or TYF-MS trnsfected cells (p>.79). On the right pnel, Fluc RNA levels were first normlized to cyc RNA levels. The rtio of reltive Fluc RNA levels in pellet frction to those in sucrose frction ws compred etween MS nd TYF-MS trnsfected cells. p<.7 determined y Student's t test. h, S cells on -well pltes were cotrnsfected with ng of Ac/UTR-FLUC-UTR-6xBS, ng of Ac/RLUC, ng of Ac/MS-V or Ac/TYF-MS-V, nd the incresing mount ( or 7 ng) of Ac/TYF-V (or TYF deletion constructs). Dul luciferse ssys were performed t 8 h fter trnsfection. Activtion fold of FLUC reporter ws clculted similrly s ove. Results were verged from - experiments. Error rs indicte stndrd devitions (d, h) or SEM (e-g). p<. nd p<. s determined y Student s t test. AlldChoe Supplementry Figure

AA(A)n NUCLEUS CLK CYC AA(A)n trnsltionlly ctive R

P PER P TIM DBT PER P TIM DBT PER TIM Supplementry

Newly synthesized per trnscript ssocites with

temporrily repress their trnsltion, cusing

TYF, which does not hve known RNA recognition

stimulte trnsltionl initition y susequent

15 doi:.8/nture978? CYTOPLASM PABP trnsltionlly inctive AA(A)n PABP R AA(A)n NUCLEUS CLK CYC AA(A)n trnsltionlly ctive R per, tim? P PER P TIM DBT PER P TIM DBT PER TIM Supplementry Figure. A model for TYF function in PER expression. Newly synthesized per trnscript ssocites with RNA-inding posttrnscriptionl regultors (red R) tht temporrily repress their trnsltion, cusing trnsltionl dely in PER synthesis. TYF, which does not hve known RNA recognition motif, could e then recruited to trget trnscripts y these trnsltionl repressors. TYF ssocition could relese these repressors to stimulte trnsltionl initition y susequent interctions with the eife-contining cp-inding complex (eiff) nd PABP. TYF function might lso e especilly importnt on poorly denylted trget trnscripts. AlldChoe Supplementry Figure

16 doi:.8/nture978 Supplementry Tle. tyf hypomorphic muttions cuse wek ut long period rhythm Genotype n %R Period +/- SEM Power +/- SEM c +/Y.8 +/ /-. tyf /Y 6.6 +/ /-.8 tyf e /Y / /-. tyf e #/Y d / /-. tyf e #/Y d. +/ /- 8. tyf e #/Y d. +/ /- 7.6 tyf e #/Y d. +/ /- 8. tyf e #/Y d 9. +/-.. +/- 7.6 tyf e #6/Y d. +/ /- 6. tyf e #8/Y d / /- 8. tyf e #9/Y d 97. +/ /- 9. tyf e #/Y d 97. +/ /-.8 tyf e #/Y d. +/ / /+ 9. +/ /- 7.8 tyf / /-..8 +/-.6 tyf e / / /-. tyf /tyf.9 +/-.9. +/-. tyf e /tyf e /-.. +/-.8 tyf e /tyf /-.. +/-. Df()r/+ 8. +/-..7 +/- 7. Df()r/tyf 6. +/ /-. Df()r/tyf e /-.. +/-.9 Df()HC/+ 9. +/ /-.6 Df()HC/tyf / /-. Df()HC/tyf e /-.. +/- 6. n indictes numer of flies nlyzed %R indictes percent flies with detectle rhythmicity (P-S>) c Power is mesure of rhythmic strength d precise excision lines from tyf e llele AlldChoe Supplementry Tle 6

17 doi:.8/nture978 Supplementry Tle. TYF overexpression lengthens free-running locomotor rhythm Genotype n %R Period +/- SEM Power +/- SEM c tim-gal/ / /-. Pdf-GAL/+.8 +/ /- 7.9 tyf G87 /Y / /-. tyf G87 /Y; tim-gal/ / /-. tyf G87 /Y; tim-gal/+; cry-gal8/+. +/ /- 9.6 tyf G87 /Y; tim-gal/pdf-gal /-.. +/-. tyf G87 /Y; Pdf-GAL/ /-.. +/- 6. UAS-mGFP/+ 6. +/ /- 9. UAS-tyf #/+ 6. +/ /-.7 UAS-tyf #6/+ 7. +/ /- 7.6 UAS-tyf #7/+. +/-.. +/- 8. UAS-tyf #8/+. +/ /- 8. UAS-tyf #9/+. +/-.. +/-. UAS-tyf #/ / /- 8. tim-gal/ / /-.6 tim-gal/+; UAS-mGFP/ / /- 7. tim-gal/+; UAS-tyf #/+. +/ /- 7.9 tim-gal/uas-tyf #6 6. +/ /- 7. tim-gal/uas-tyf # / /-.9 tim-gal/uas-tyf #8. +/-..6 +/- 6.9 tim-gal/+; UAS-tyf #9/+ 6. +/ /- 8.6 tim-gal/+; UAS-tyf #/ / /- 9. Pdf-GAL/+. +/ /- 6.8 Pdf-GAL/+; UAS-mGFP/+. +/-..7 +/- 8. Pdf-GAL/+; UAS-tyf #/+ 6. +/-.. +/- 9. Pdf-GAL/UAS-tyf # /-..8 +/- 6. Pdf-GAL/UAS-tyf # /-..8 +/- 7. Pdf-GAL/UAS-tyf #8.8 +/ /- 6.8 Pdf-GAL/+; UAS-tyf #9/ /-.. +/- 9.8 Pdf-GAL/+; UAS-tyf #/ / /-.9 n indictes numer of flies nlyzed %R indictes percent flies with detectle rhythmicity (P-S>) c Power is mesure of rhythmic strength AlldChoe Supplementry Tle 7

18 doi:.8/nture978 Supplementry Tle. tyf expression in PDF+ cells is necessry for wild-type locomotor rhythm Genotype n %R Period +/- SEM Power +/- SEM c tyf Δ /Y; tim-gal/ /-.7. +/-.7 tyf Δ /Y; tim-gal/+; UAS-mGFP/ /-.6. +/-. tyf Δ /Y; tim-gal/+; UAS-tyf #/+. +/ /- 6. tyf Δ /Y; tim-gal/uas-tyf # / /-.8 tyf Δ /Y; tim-gal/uas-tyf # / /- 7. tyf Δ /Y; Pdf-GAL/+ 6. +/ /-. tyf Δ /Y; Pdf-GAL/+; UAS-mGFP/+ 6. +/-.. +/-. tyf Δ /Y; Pdf-GAL/+; UAS-tyf #/ / /- 8.9 tyf Δ /Y; Pdf-GAL/UAS-tyf #6 8. +/ /- 7.7 tyf Δ /Y; Pdf-GAL/UAS-tyf #7 9. +/ /- 8. tyf e /Y; tim-gal/ / /-.9 tyf e /Y; tim-gal/+; UAS-mGFP/ /-..6 +/-. tyf e /Y; tim-gal/+; UAS-tyf #/ / /- 7. tyf e /Y; tim-gal/uas-tyf # / /- 7.6 tyf e /Y; tim-gal/uas-tyf #7. +/ /- 7. tyf e /Y; Pdf-GAL/ / /-. tyf e /Y; Pdf-GAL/+; UAS-mGFP/ /-.8. +/-.7 tyf e /Y; Pdf-GAL/+; UAS-tyf #/+ 8. +/ /-. tyf e /Y; Pdf-GAL/UAS-tyf # /-.. +/- 8.9 tyf e /Y; Pdf-GAL/UAS-tyf # / /- 7. Pdf-GAL/ / /- 6. UAS-tyfRNAi / /- 6.6 Pdf-GAL/+; UAS-tyfRNAi.9 +/ /-. n indictes numer of flies nlyzed %R indictes percent flies with detectle rhythmicity (P-S>) c Power is mesure of rhythmic strength AlldChoe Supplementry Tle 8

19 doi:.8/nture978 Supplementry Tle. Adult-specific TYF overexpression rescues circdin phenotype in tyf mutnt flies Genotype n RU86 %R c Period +/- SEM Power +/- SEM d +/Y -. +/-..6 +/-. +/Y / /- 7. +/Y; UAS-tyf-V/+; ElvGS-GAL/+ -. +/-..7 +/- 8. +/Y; UAS-tyf-V/+; ElvGS-GAL/ /-..7 +/- 9. +/Y;; ElvGS-GAL/UAS-tyf # -.8 +/ /-. +/Y;; ElvGS-GAL/UAS-tyf # +.6 +/-..9 +/- 8.8 tyf /Y / /-.8 tyf /Y +.7 +/-..7 +/-. tyf /Y; UAS-tyf-V/+; ElvGS-GAL/ / /-. tyf /Y; UAS-tyf-V/+; ElvGS-GAL/ / /-.8 tyf /Y;; ElvGS-GAL/UAS-tyf # / /- 8.6 tyf /Y;; ElvGS-GAL/UAS-tyf # / /-. tyf e /Y /-..6 +/-.7 tyf e /Y / /-. tyf e /Y; UAS-tyf-V/+; ElvGS-GAL/ /-.. +/-.6 tyf e /Y; UAS-tyf-V/+; ElvGS-GAL/ / /- 8. tyf e /Y;; ElvGS-GAL/UAS-tyf # /-.7. +/-. tyf e /Y;; ElvGS-GAL/UAS-tyf # / /- 9. n indictes numer of flies nlyzed fly foods in the ehvior test contined either (+). mm RU86 or (-) % ethnol s vescle control. c %R indictes percent flies with detectle rhythmicity (P-S>) d Power is mesure of rhythmic strength AlldChoe Supplementry Tle 9

20 doi:.8/nture978 Supplementry Tle. PER overexpression rescues circdin phenotype in tyf mutnt flies Genotype n %R Period +/- SEM Power +/- SEM c Pdf-GAL/+. +/ /- 6.8 Pdf-GAL/+; UAS-mGFP/+. +/-..7 +/- 8. Pdf-GAL/+; UAS-per6/+. +/-.. +/-. Pdf-GAL/UAS-tim.7 +/-..9 +/- 8.6 Pdf-GAL/+; UAS-Clk/+. +/-..9 +/-.8 Pdf-GAL/UAS-tyf-V 6.6 +/-..7 +/- 7.8 tyf Δ ; Pdf-GAL/+ 6. +/ /-. tyf Δ ; Pdf-GAL/+; UAS-mGFP/+ 6. +/-.. +/-. tyf Δ ; Pdf-GAL/+; UAS-per6/ / /-.7 tyf Δ ; Pdf-GAL/UAS-tim 7. +/-.8. +/-. tyf Δ ; Pdf-GAL/+; UAS-Clk/+ 8. +/ /-. tyf Δ ; Pdf-GAL/UAS-tyf-V 9.8 +/-.. +/-. tyf e ; Pdf-GAL/ / /-. tyf e ; Pdf-GAL/+; UAS-mGFP/ /-.8. +/-.7 tyf e ; Pdf-GAL/+; UAS-per6/+. +/ /-.7 tyf e ; Pdf-GAL/UAS-tim 6. +/ /-. tyf e ; Pdf-GAL/+; UAS-Clk/ / /-. tyf e ; Pdf-GAL/UAS-tyf-V 96. +/ /-. n indictes numer of flies nlyzed %R indictes percent flies with detectle rhythmicity (P-S>) c Power is mesure of rhythmic strength AlldChoe Supplementry Tle

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI:.8/NPLANTS.6. Results A homozygous muttion in cls- results in incomplete cytokinesis nd seedling-lethl phenotypes, wheres heterozygous mutnt plnts develop normlly. This suggests tht the cls- llele