SUPPLEMENTARY INFORMATION

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1 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION SPPLEMENTRY FIRE LEENDS for Okmr et l, "The hirpin RN pthwy enertes endoenos sirns in Drosophil" Fire S. Smll RN size nlysis effetively distinishes between bon fide hirpin trnsripts nd fortitos inverted repets omprised of trnsriptionlly onverent or diverent ene dplites. () EINVERTED hit ontinin diverently oriented trypsin enes enertes mny smll RNs; ble hits re on the (-) strnd nd red hits re on the (+) strnd. (B) Size nlysis shows brod rne of strnd-seletive, probble derdtion frments, sestin tht there is not sinle trnsript tht trverses this reion. () EINVERTED hit representin novel tndem pir of short hirpins tht we provisionlly nmed ho39- nd ho39-2. (D) Size nlysis shows tht predominntly 22 nt RNs re proded from these loi. lthoh these re mirn-like, the short terminl loop sests tht they re not onventionl mirns. Nevertheless, they pper to hve been proded by some type of hirpin prersor. (E) EINVERTED hit ontined within the 8854 ene nnottion; the vst mjority of reds re from the (-) strnd. (F) Size nlysis shows tiht distribtion of smll RNs entered rond 2 nt. Fire S2. Detils of hp () Smll RNs were loned predominntly from the 5' TR nd 3' TR of the nnotted 8854, bt its odin potentil is limited. enome view ws provided by S ( (B) lones mppin to the 3' TR lster exhibit ler phsin, lthoh it is not s preise s with hp-4068 (Fi. B, Fi. S3). () The 5' TR nd 3' TR lsters re omplementry nd form n extended dplex. (D) losep of the dominnt lones mpped to one sement of the dplex illstrtes lerly phsed dplexes with ~2nt-3' overhns.

2 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION Fire S3. Detils of hp () series of 20 tndem repets re fond between 6903 nd loned smll RNs mp to the (-) strnd, nd pper to initite in the 3' TR of The extent of the ' TR hs been onfirmed by DN lones. ny individl repet forms hirpin (B), while ny pir of repets forms loner hirpin (); even loner hirpins involvin dditionl repets re possible. The seqenes nd strtres of the first nd the first+seond hirpins in the tndem rry re depited. (D) Most of the hp-4068 lones mp to mny lotions (p to 20), bt the most distlly-mppin lones orrespond to niqe lotions in the ' TR. Fire S4. Detils of hprn. olletion of seeminly heteroenos reds mpped to this hirpin (s smmrized on the left shemti); the thikness of the br is proportionl to the nmber of reds. However, seretion of the reen-hihlihted lones reveled preferred leve reister yieldin sirn dplexes (middle shemti). pprent phsin of the yellow-hihlihted lones potentilly reflets n lterntive leve reister (riht shemti). Fire S5. Detils of hp-pnr009. One mjor sirn dplex with nonil 2nt-3' overhns old be mpped to its hirpin; some of these reds lso mp to hp nd hp-r nother reion with niqely mppin reds exhibited phsin (i.e., djent-mppin reds). These nd other smll RN reds tht mp niqely to this hirpin provided evidene for its independent expression nd proessin. dditionl rre lones re not depited. Fire S6. Detils of hp Two mjor sirn dplexes old be mpped to its hirpin; some of these reds lso mp to either hp-pnr009 or hp-r However, one of these sirn reds mpped niqely to this los. These nd other smll RN 2

3 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION reds tht mp niqely to this hirpin provided evidene for its independent expression nd proessin. dditionl rre lones re not depited. Fire S7. Detils of hp-r Two mjor sirn dplexes old be mpped to its hirpin; these reds lso mp to either hp nd pnr009. These nd other smll RN reds tht mp niqely to this hirpin provided evidene for its independent expression nd proessin. dditionl rre lones re not depited. Fire S8. The pnr009, hp-32207, hp-r32205 loi re lstered within 70kb of eh other in the hr3l:9,47,000-9,482,000 reion. The R32205 nd pnr009 enes were previosly nnotted s pttive non-odin RNs. s seen in Fis. S5-S7, some reds mp to two or three of these hprn loi. The shemti ws enerted sin browse t Flybse ( Fire S9. Detils of n hprn ndidte downstrem of koi. It ontins n extended dplex sement tht is in the size rne of hprns (nd lrer thn tht of typil premirns). Its loop reion inldes repetin element tht mthes to over 50 lotions. However, its stem reion is omposed of niqe seqene, nd enertes smll nmber of loned smll RNs. The bse of the stem enertes eiht 2nt RNs while the top of the stem enertes 25 nt nd 26 nt RN. While it mthes minimlly to or hirpin riteri, the pttive sirns were not rrned s typil sirn dplex. Therefore, we tenttively desinte this ndidte hprn los. Perhps its leve pttern miht beome lerer with dditionl lones, s with hprn, for whih the vilble lones potentilly sest dl leve ptterns. 3

4 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION Fire S0. Bioenesis nd strtrl nlysis of hprns. () Northern nlysis of hp in S2 ells treted with vrios dsrns. Its bioenesis reqirements re similr to other hprn-derived sirns, in tht its mltion is most dependent on Loqs, Dr-2 nd O2 (boxed reions). Note lso tht ldderin is inded in the O2-knokdown ells; minor mont of ldderin is lso deteted in Dr-2- knokdown ells. (B) 5' nd 3' end nlysis of smll RNs. Bntm mirtes more qikly followin ß-elimintion of 3' ends, nd its levels pper to be reded s well. onversely, bntm mirtes more slowly followin IP tretment to remove 5' phosphte. hp-8854 nd hp-4068 re resistnt to ß-elimintion, bt sensitive to IP. Fire S. The 3' TR of 4068 does not pper to be tret of hp () sensor ontinin the 'TR ws not repressed in the presene of vrios hp expression onstrts. (B) The ' TR sensor ws not de-repressed in the presene of vrios SOs. Fire S2. Derepression of ms308 in Dr2- nd o2-defiient ells. S2 ells were treted with dsrn inst FP, Dr-2 or o2. qpr ws sed to mesre ms308 level in eh ondition, s normlized to rp49 level. The rph depits the ms308 level in Dr-2- nd o2-depleted ells, reltive to FP dsrn-treted ells. These dt were pooled from six experiments; stndrd devitions re shown. Fire S my be tret of hp () hp-8854 is derived from These enes re loted on different hromosomes, bt shre sinifint similrity. (B) hp-8854 is not simple inverted repet of 8289, however, s 4

5 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION portions of the 8289 seqene re trnsposed with respet to eh other in hp () Etopi hp-8854 speifilly repressed 8289:FP fsion. 5

6 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION derdtion RNs hr2r:7,235,869-7,240,000 EINVERTED hit mm-trypsin 3003 (-) strnd reds (+) strnd reds smll RN red ont b nt lenth novel short hirpins hrx:9,452,369-9,452,794 d EINVERTED hit ho39- (+) strnd reds ho left-rm smll RN riht-rm smll RN ho39-...((((.(((.((((.((((((((.(((.((((((...)))))).))).)))))))).)))).))).))))... (-30.4) ho39-2 (((...((((.(((.((((.((((((((.(((.((((((...)))))).))).)))))))).)))).))).))))...)))... (-30.5) e hirpin RNs hr2l:9,788,600-9,790,490 EINVERTED hit (+) strnd reds (-) strnd reds 8854 smll RN red ont f nt lenth Okmr et l, Spplementry Fi. 6

7 8854 5'TR lster 'TR lster 'TR lster smll RN phsin nt loop (fter spliin) 'TR lster 'TR lster Okmr et l, Spplementry Fi. 2 b d red 234 reds 44 reds 7 reds 0 reds 95 reds 25 reds B red hp-8854 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION 7

8 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION probe B probe probe D probe 2 reds 2 reds 35 reds 409 reds 62 reds 46 reds 5 reds 45 reds 94 nt loop repet 2 reds 35 reds 409 reds 62 reds 46 reds 5 reds 45 reds 2 reds probe B probe probe D probe probe B probe probe D two repet hirpin 2 reds 35 reds 409 reds 62 reds repet 46 reds 5 reds 45 reds 2 reds probe hp-4068 b one repet hirpin } smll RN reds d niqe lones enerted from the 5 -most hprn tndem rry 20 BLST hits BLST hit Okmr et l, Spplementry Fi

9 hprn hr3r:5,746,45-5,746,598 (-) (downstrem of 4770) nlbeled brs represent sinle lones leve reister leve reister 2? Okmr et l, Spplementry Fi. 4 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION 9

10 d = hp-pnr009 hr3l:9,479,846-9,48,488 (-) 9 reds red mjor sirn dplex phsin 26 reds, lso mthes hp nd hp-r reds, lso mthes hp-r32205 (bt not hp-32207) reds, lso mthes hp nd hp-r32205 Okmr et l, Spplementry Fi. 5 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION 0

11 d = hp-32207, nt reds 5 reds, lso mthes hp-pnr009 nd hp-r reds, lso mthes hp-r32205 (bt not hp-pnr009) 26 reds, lso mthes hp-pnr009 nd hp-r32205 mjor sirn dplex minor sirn dplex phsed lones red, lso mthes hp-r32205 (bt not hp-pnr009) 2 reds, lso mthes hp-pnr009 nd hp-r32205 Okmr et l, Spplementry Fi. 6 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION

12 d = hp-r reds, lso mthes hp-pnr009 nd hp reds, lso mthes hp-pnr009 (bt not 32207) 5 reds, lso mthes hp-pnr009 nd hp reds, lso mthes nd hp (bt not hp-pnr009) Okmr et l, Spplementry Fi. 7 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION 2

13 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION three pnr009-fmily hprn loi in the hr3l:9,400,000-9,500,000 reion ene Spn 3 mrn 2 DS Non odin RN Ntrl trnsposon Okmr et l, Spplementry Fi

14 downstem of koi hr2r:2,704,745-2,705,362 (+) EINVERTED with lones pirn sized lones repetin element ~50 BLST 5 2 pirn sized niqely-mpped lones sirn sized niqely-mpped lones Okmr et l, Spplementry Fi. 9 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION 4

15 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION dsrn: FP Drosh Psh Dr- Loqs Dr-2 R2D2 Exp5-N Exp5- O O hp mtre sirn 20 b None β-elim IP 30 bntm 20 hp hp Okmr et l, Spplementry Fi

16 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION pdsred pdsred-hp-4068 ( repet) pdsred-hp-4068 (2 repets) b SO-hp-8854B SO-hp-4068B SO-hp Normlized sensor tivity Normlized sensor tivity psihek psihek TR 0 psihek psihek TR Okmr et l, Spplementry Fi. 6

17 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION fold expression reltive to FP dsrn FP dsrn Dr-2 dsrn O2 dsrn 0 ms308 Okmr et l, Spplementry Fi

18 doi: 0.038/ntre0705 SPPLEMENTRY INFORMTION hrx:6b kb 5 TR hr2l:30d sirn lster 3 TR sirn lster 8854 kb // // IP3K b 8289 B hp-8854 B B' ' ' 250nt tb-fp tb-8289::fp DsRed +hp-8854 () +hp-8854 () +hp-4068 α-fp α-tblin α-fp α-tblin Okmr et l, Spplementry Fi