Bidirectional processing of pri-mirnas with branched terminal loops by Arabidopsis Dicer-like1

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1 Bidiretionl proessing of pri-mirnas with rnhed terminl loops y Aridopsis Dier-like Hongling Zhu 3, Yuyi Zhou,2,4, Cludi Cstillo-González,2, Amer Lu,2, Chunxio Ge 2,5, Ying-To Zho 6, Liusheng Dun 4, Zhohu Li 4, Mihel J Axtell 7,8, Xiu-Jie Wng 6 & Xiuren Zhng,2,5 3 Nture Ameri, In. All rights reserved. MiroRNAs (mirnas) originte from primry trnsripts (pri-mirnas) with hrteristi stem-loop strutures, nd their urte proessing is required for the prodution of funtionl mirnas. Here, using the pri- fmily in Aridopsis thlin s prdigm, we report the ruil role of pri-mirna terminl loops in mirna iogenesis. We found tht multirnhed terminl loops in pri-s sustntilly suppress expression in vivo. Unlike nonil proessing of pri-mirnas, terminl loop rnhed pri-mirnas n e proessed y Dier-like () omplexes idiretionlly from se to loop nd from loop to se, resulting in produtive nd ortive proessing of mirnas, respetively. In oth ses, omplexes nonilly ut pri-mirnas t distne of 6 7 p from referene single-strnded loop region. lso djusts proessing sites towrd n internl loop through its helise domin. These results provide new insight into the poorly understood proessing mehnism of pri-mirnas with omplex seondry strutures. mirna iogenesis strts with the trnsription of long pri-mirnas, typilly y RNA polymerse II. pri-mirnas re hrterized y stem-loop strutures onsisting of terminl loop, n upper stem, duplex of mirna nd its omplementry strnd (mirna: mirna), lower stem nd flnking single-strnded sl segments (Fig. ). In nimls, pri-mirnas re first proessed y n RNse III enzyme, Drosh, nd its prtner, DGCR8 protein (lso known s Psh). DGCR8 ssoites with the se of the stem-loop struture nd sets the tlyti site of Drosh p wy from the singlestrnded RNA (ssrna) doule-strnded RNA (dsrna) juntion 2. The resultnt produts, known s preursor mirnas (pre-mirnas), re further leved y nother RNse III enzyme, Dier, to relese ~2-p mirna:mirna duplexes 2. Mture mirnas re loded into Argonute (AGO)-ontining RNA-indued silening omplexes nd guide AGO proteins to repress their omplementry trgets,3 0. In Aridopsis,, one of four Dier homologs, orhestrtes this entire proess, inluding the onversion of pri-mirna to pre-mirna nd the relese of pre-mirna to mirna:mirna duplexes 5,. hs two oftors, serrte (SE) nd dsrna-inding protein lled hyponsti leves (HYL), mong others 2. HYL nd SE hve een proposed to hve role nlogous to tht of DGCR8 y ting s moleulr ruler for mirna proessing in plnts 3. Plnt primirna hirpins re heterogeneous in length nd struture, with vrile positioning of the mirna:mirna duplex 2,22. Geneti studies hve demonstrted tht n imperfetly pired lower stem of ~5 p elow the mirna:mirna duplex is key element for the initil pri-mirna levge, whih led to the proposed 5-nt model in whih the initil pri-mirna proessing often ours t distne of ~5 nt from either ssrna-dsrna juntions or internl unstrutured regions In ontrst, the terminl loop nd upper stem re lrgely tolernt of muttions for some pri-mirnas 24 ut not others 27,28. Moreover, mny plnt pri-mirnas hror rnhed terminl loops (BTLs) or multiple ulges or internl loops in the lower nd upper stems. How pri-mirnas with omplex seondry strutures re reognized nd proessed remins elusive. mir-65 nd re two relted mirnas tht differ in sequene y only nt nd oth trget HD-ZIP III trnsripts 29, to regulte meristem development nd orgn polrity. They re enoded y nine loi in the Aridopsis genome (MIR65, MIR65 nd MIR66 through MIR66g). pri-mir-65s nd pri-s ontin diversified strutures rnging from liner foldks to omplited rnhed loops t vrile lotions. Here we found tht BTLs mrkedly deresed undne in the mjority of ses. omplexes proessed terminl loop rnhed (TLBed) pri-mirnas idiretionlly, either nonilly from se to loop, resulting in mirna prodution, or nonnonilly from loop to se, leding to ortive proessing. omplexes leved pri-mirnas t 6 7 p wy from referene ssrna-dsrna juntions for either nonil or nonnonil proessing, lthough ould lso djust the proessing sites ner n internl loop through its helise domin. These results shed light on the omplited reltionship etween pri-mirna strutures nd mirna iogenesis in plnts. Deprtment of Biohemistry nd Biophysis, Texs A&M University, College Sttion, Texs, USA. 2 Institute of Plnt Genomis nd Biotehnology, Texs A&M University, College Sttion, Texs, USA. 3 College of Food Siene nd Nutritionl Engineering, Chin Agriulturl University, Beijing, Chin. 4 College of Agriulture nd Life Sienes, Chin Agriulturl University, Beijing, Chin. 5 Progrm of Moleulr nd Environmentl Plnt Sienes, Texs A&M University, College Sttion, Texs, USA. 6 Stte Key Lortory of Plnt Genomis, Institute of Genetis nd Developmentl Biology, Chinese Ademy of Sienes, Beijing, Chin. 7 Deprtment of Biology, Penn Stte University, University Prk, Pennsylvni, USA. 8 Huk Institutes of the Life Sienes, Penn Stte University, University Prk, Pennsylvni, USA. Correspondene should e ddressed to X.Z. (xiuren.zhng@tmu.edu). Reeived 4 Jnury; epted 8 July; pulished online August 3; doi:0.038/nsm.2646 nture struturl & moleulr iology dvne online pulition

2 3 Nture Ameri, In. All rights reserved. RESULTS BTLs of pri-mirnas repress mirna umultion in vivo We reently oserved tht trnsgeni plnts hroring different pri-mir-65s nd pri-s, even though they re expressed under the sme onstitutive 35S promoter, showed diverse phenotypi normlities rnging from severe pinhed, rdil or urled leves to nerly wild-type phenotypes (Supplementry Fig.,). This phenotypi severity is proportionl to the mounts of mir-65 nd (Fig.,). Mfold nlysis of the pri-mirnas 3 reveled two mjor types of seondry strutures: one is roughly liner foldk, nd the other hrors BTLs. Notly, pri-mir-65s nd pri-s with liner hirpins generlly showed stronger morphologil defets nd more undnt mirna umultion thn those with BTLs, exept for pri-mir-65 nd pri- (Fig.,). These oservtions indite tht the seondry strutures of pri-mirnas hve deisive role in mirna undne. pri- nd pri-f showed the lowest nd highest levels of, respetively. Both of these pri-mirnas ontin two internl loops nd one ulge in their lower stems. However, pri- hs BTLs, wheres pri-f hrors less strutured terminl region (Fig. ). To delinete the struturl determinnts tht ffet mirna expression, we onduted se-top swpping experiments etween pri- nd pri-f. Swithing either the se or top of pri- to pri-f sustntilly deresed umultion nd ttenuted the trnsgeni phenotype (Fig. d nd Empty vetor MIR65 MIR65 mir-65 Empty vetor MIR66 MIR66 MIR66 MIR66d 2 Terminl loop Upper stem Lower stem mirna duplex Bsl segment mirna mirna Supplementry Fig. ). Conversely, swpping of the pri-f top to pri- led to enhned umultion nd more severe phenotypes. Notly, we did not oserve this enhnement with trnsgeni plnts expressing himeri pri- f B, whih hrors the se (B) of pri-f (f) in the pri- ontext (Fig. e nd Supplementry Fig. d). These results were reproduile with oth the extended nd onise forms of pri-mirnas in Niotin enthmin (Supplementry Fig.,f,g). Thus, omintion of se, top nd mirna:mirna duplex in n pproprite pri-mirna ontext is required for mximum mirna umultion. pri- BTLs ontin two smll loe loops (leled nd 2) tthed to lrge terminl loop (leled 3) (Fig. f, MIR66 top). Deletion of loop 3 (MIR66 T nd MIR66 T2), redution in the size of loop 3 (MIR66 Ts3) nd lineriztion of the foldk (MIR66 T2+3) ll sustntilly inresed umultion nd enhned the severity of the trnsgeni phenotypes, wheres the lrge terminl loop in the top region (MIR66 T3) did not hve n oservle effet reltive to pri- (Fig. f nd Supplementry Fig. e). We onlude tht the size, shpe nd distne of terminl loops reltive to mirna:mirna duplexes n e ruil for mirna umultion in vivo. of pri- lowers levels pri- nd pri-f hd omprle primry trnsript levels in vivo (Supplementry Fig. 2), result inditing tht the MIR66e MIR66f MIR66g d Empty vetor MIR66f B MIR66f T mir66f MIR66f mir66 MIR66f T MIR66f B MIR66f B+T mir66 MIR66 mir66 mir-64 mir-64 mir-64 Figure The seondry strutures of pri-mirnas ffet mirna undne in vivo. () Shemti struture of representtive pri-mirna. (,) srna lot nlysis of mir-65 nd in T trnsgeni plnts overexpressing nine MIR65 nd MIR66 fmily memers. The predited seondry strutures of the pri-mir-65s nd pri-s re shown ove. mirna nd mirna re shown in turquoise nd red, respetively. Totl RNA ws prepred from pool of T trnsformnts (n > 0 for eh onstrut). srna lots were proed using -end 32 P-leled oligonuleotide proes omplementry to the indited mirnas (Supplementry Tle ). mir-64 ws the loding ontrol. (d,e) srna lot nlysis of, performed s in nd, in T stle trnsformnts overexpressing deletion ( ) or element-swpped mutnts of pri-f (d) nd pri- (e). e Empty vetor MIR66 B B MIR66 T mir66 MIR66 mir66 The ses (B), mirna:mirna duplexes nd tops (T) of pri- nd pri-f re shown in different olors in the shemti strutures. (f) srna lot nlysis of, performed s in nd, in T stle trnsformnts overexpressing MIR66 with vrious deletions in the terminl loop. Shemti strutures of the terminl regions of pri- deletion mutnts re shown ove. Loops, 2 nd 3 re shown in pink, greenish yellow nd mroon, respetively. The orresponding unropped imges re shown in Supplementry Figure 9. MIR66 f T MIR66 f B MIR66 f B+T mir66f MIR66f mir66 mir-64 f Empty vetor MIR66 top 3 MIR66 T MIR66 T2 MIR66 T3 MIR66 T2+3 MIR66 T s3 mir-64 dvne online pulition nture struturl & moleulr iology

3 3 Nture Ameri, In. All rights reserved. pri-f pri-f -endleling -endleling Internl leling 0 0 min Lne f f3 (89 nt) f4 (47 nt) f2 (35 nt) f (26 nt) f5 (22 nt) f5 nd f6 (2 nt) differentil umultion of in their trnsformnts ws regulted t post-trnsriptionl level. We hypothesized tht different seondry strutures of pri- nd pri-f my lter their proessing effiieny nd/or ury y omplexes. To test this hypothesis, we developed n in vitro system of reonstitution ssys (Supplementry Fig. 2 d). We oexpressed 35S-driven tgged with two Flg nd four My epitopes (2Flg- 4My-), HYL tgged with six My epitopes (6My-HYL) nd SE tgged with three hemgglutinins (SE-3HA) in N. enthmin euse HYL SE omplexes n form n effiient mirna proessor 5,32. The purified omplexes leved the -endleled pri-f trnsript t the expeted position, generting primrily 26-nt frgment, whih is the -flnking segment (f; 26 nt) (Fig. 2,). We performed prllel experiment with internlly leled pri-f nd oserved six mjor frgments: f, the -flnking segment (f2; 35 nt), pre-f (f3; 89 nt), the upper stem nd terminl loop (f4; 47 nt) nd : duplexes (f5 nd f6; ~2 22 nt). Hene, the in vitro reonstitution ssys were le to repitulte the proess of mirna iogenesis in vivo. In shrp ontrst to the pri-f proessing, inution of -endleled pri- with omplexes generted equl mounts of two mjor frgments (Fig. 2,d). One of these frgments (; 24 nt) yielded funtionl pre-mirna, representing produtive proessing. However, the other frgment ( ; nt) suggested norml, ortive levge in pri- euse the leved produt did not ontin the entire sequene. We further onfirmed the presene of two proessing ptterns for pri- with internlly leled pri- Figure 2 The onomitnt presene of produtive nd ortive proessing of pri- ut not pri-f. () Predited seondry struture of in vitro trnsried pri-f. Red rrowheds indite the expeted initil proessed sites of pri-f. () In vitro reonstitution ssy with -end-leled or internlly leled pri-f trnsripts. Immunopreipittes were prepred through two-step ffinity f f2 f5 f6 f3 f4 pri- d pri- Internl leling 0 min Lne e (p) Lne 2 3 Mrker Col-0 35S-MIR66 3 (06 nt) (74 nt) 4 (64 nt) ( nt) 2 (38 nt) (24 nt) 2 (22 nt) 5 nd 6 (2 nt) /7 5/7 2 6/7 3/7 /7 /7 trnsripts, whih yielded two sets of levge frgments: 6 frgments, whih re equivlent to f f6 from pri-f desried ove nd whih resulted from onstrutive proessing, nd 3 frgments, whih were derived from ortive proessing. To determine whether ortive proessing of pri- ws present in plnt, we performed experiments of rpid mplifition of DNA ends ( RACE). Although we did not reover ler intermedite proessing produts from pri- in wild-type plnts, we identified distint proessing intermedites from 35S-MIR66 trnsgeni plnts (Fig. 2e). These proessing produts indeed orresponded lrgely to the levge positions tht we found in our in vitro nlysis, inditing tht ortive proessing of pri- proly ounts for the lower umultion of from pri- in vivo (Fig. 2d nd Supplementry Fig. 3 e). Beuse we were unle to reover SE in our purified HYL omplexes, we repeted the ssys ut dded purified SE (Supplementry Fig. 2d f). The ddition of SE hd no ovious effet on the levge pttern or ury for pri- or pri-f, result suggesting tht this protein might not diretly ontriute to the proessing of pri-mirnas. In greement with this notion, reent work hs demonstrted tht SE funtions s sffold for C-terminl domin phosphtse-like (CPL) to dephosphorylte HYL, therey modulting its tivity 33. proesses pri- in opposite diretions The presene of two sets of pri- proessing produts suggests tht the -entered miroproessor reognizes purifition using ntiodies to Flg nd then ntiodies to My from N. enthmin expressing 2Flg-4My-, 6My-HYL nd SE-3HA omplexes () nd were prepred from mok-infiltrted plnts., ontrol hek. RNAs reovered from the retion mix were frtionted on 5% denturing gels nd deteted y phosphor imging. The positions of intt sustrtes, levge produts nd retion times re indited. Right, shemti illustrtion of the intermedite nd finl levge produts (f f6) of pri-f y. () Predited seondry struture of in vitro trnsried pri-. Red nd turquoise rrowheds show the expeted initil produtive proessing nd the oserved ortive proessing sites of pri-, respetively. (d) In vitro reonstitution ssys with -end-leled or internlly leled pri- trnsripts. The ssys were onduted s in. Right, shemti illustrtion of the intermedite nd finl levge produts ( 6 nd ) of pri- y. (e) Produtive nd ortive proessing frgments s deteted y RACE experiments in trnsgeni plnts expressing 35S-MIR66 in vivo. The PCR produts from the RACE experiments were reovered, sequened nd mpped on pri-, nd their ounts re shown on the right. Col-0 is wild-type Aridopsis thlin eotype. The orresponding unropped imges re shown in Supplementry Figure 9. 2 nture struturl & moleulr iology dvne online pulition

4 3 Nture Ameri, In. All rights reserved. E7Q E696Q E7Q E696Q ( H) NLS Lys243 RNA helise Al764 DUF PAZ RNse III dsrbd Figure 3 omplexes proess pri- idiretionlly from lower stem to loop nd from loop to lower stem. () Shemti illustrtion of the domins nd mutnts.,, nd ( H) refer to RNse III (E7Q) nd III (E696Q) point muttions nd doule muttions (E7Q E696Q) nd helise-domin deletion mutnt, respetively. () In vitro levge ssys with -end-leled pri- trnsripts y nd the mutnts. Immunopreipittion, levge ssys nd RNA proessing were performed s in Figure 2. Blk sterisks show residul nonspeifi levge tht is frequently present in the ssy system. pri- two lotions in pri- for levge. To understnd how omplexes reognize pri-, we modified to exmine the inding orienttion of omplexes reltive to pri-. Diers ontin n RNA helise domin, PAZ domin, one or two dsrna-inding domins nd two RNse III ( nd ) domins tht ut dsrnas onomitntly (Fig. 3). Bioinformtis nlysis reveled tht residues Glu7 nd Glu696 of re highly onserved in the nd domins of Diers mong eukryoti orgnisms 34. We generted two point muttions in, E7Q nd E696Q, whih intivte the nd domins, respetively (Supplementry Fig. 4). We resoned tht inution of these semitive mutnts with pri- my produe prtilly proessed frgments, whih ould e used to dedue the inding orienttions of in pri. Wheres (E7Q E696Q) olished RNse III tivity ompletely (Fig. 3,, lne 3), (E7Q) generted (24 nt) nd + (4 nt) frgments when we used -end-leled pri- in the ssy (Fig. 3, lne 5). In prllel, produed two frgments orresponding to the 2 (39 nt) nd 2+3 (29 nt) segments when we used -end-leled pri s sustrte (Fig. 3, lne 5). These results indite tht ut the rm of the lower stem in produtive proessing nd the rm (the site of ) of pri- in ortive proessing. Conversely, (E696Q) generted ( nt) nd +3 ( nt) frgments when we used -end-leled pri (Fig. 3, lne 4), wheres it yielded 2 (23 nt) nd 2 + (3 nt) frgments when we used -end-leled pri- Lne end-leling pri- DCL Lne end-leling + 3 ( nt) + (4 nt) ( nt) (24 nt) (29 nt) 2 + (3 nt) 2 (39 nt) 2 (23 nt) 2 RIlI RIlI 2 RIlI RIlI RIlI RII pri- Aortive proessing Produtive proessing (Fig. 3, lne 4). These results indite tht ut the rm of the lower stem in produtive proessing nd the rm of pri- (the site of ) in ortive proessing. In ddition, wild-type lso generted intermedite levge frgments (Fig. 3,, lne 2, +3 nd +), result inditing imperfetly oordinted proessing t oth sites. Tken together, these dt suggest tht hs two opposite inding orienttions in pri- (Fig. 3d): one orienttion proesses pri- through its lower stem t the se, resulting in onstrutive prodution of pre-mirnas, nd the other orienttion lunhes pri-mir- 66 through BTLs, leding to the destrution of pre-mirnas. The helise domin of modultes its tlyti tivity 32. To study whether the helise domin hs role in pri- proessing, we repeted the levge ssys using trunted with deletion of the helise domin. We found tht the deletion of the helise domin ompletely olished tivity for ortive proessing nd ompromised produtive proessing of pri- to some extent (Fig. 3e). Consistent with this result, ATP is required for ortive proessing ut is only prtilly neessry for produtive proessing (Supplementry Fig. 4). Thus, the helisemedited unwinding of pri- strutures, whih is driven y ATP hydrolysis, is required for ortive proessing. proesses pri-mirnas 6 7 p from referene site Given the idiretionl tivity of omplexes, we investigted how the levge sites re determined. Multiple internl loops or ulges present in the lower stems of pri- nd pri-f d e H Lne RIlI Right, shemti illustrtion of the levge produts. The red sterisks on the trnsripts indite the 32 P-leling positions. Red nd turquoise rrowheds show the produtive nd ortive proessing sites, respetively. () In vitro levge ssys with the -end-leled pri- trnsripts y nd the mutnts. The levge ssys nd shemti illustrtion re essentilly s in. The end leling dded n extr ytosine to the end of pri-, nd thus the lengths of the 2 nd 2 frgments in re nt longer thn those in Figure 2d. (d) Shemti of two orienttions of in the proessing of pri- nd the resultnt onstrutive or destrutive proessing ptterns. (e) In vitro levge ssys with the -end-leled pri- trnsripts y ( H). The levge ssys nd shemti illustrtion res in. The orresponding unropped imges re shown in Supplementry Figure 9. dvne online pulition nture struturl & moleulr iology

5 3 Nture Ameri, In. All rights reserved. F-WT F- L F-WT Lne Lne Lne C- L2 C- L F- L C-ILS C-LSA-5 C-LSA-7 C-LSA-2 C-LSA-5 C-LSA-7 C-LSA-2 2 C- L3 2 3 essentilly mimi ssrna-dsrna juntions nd thus my serve s the referene sites for guiding tlyti tivity. To investigte this possiility, we first nneled internl loop 2, whih is ~ p wy from the : duplex, in the lower stems of pri-f (Fig. 4, lne 4, F- L) nd pri- (Fig. 4, lne 4, C- L2) nd exmined how the levge ptterns were ffeted. Anneling internl loop 2 of either pri- or pri-f did not lter the predominnt levge ptterns. Thus, the length of the moleulr ruler in pri-mirna proessing in plnts is not ~ p, s hs een oserved in nimls 2. We next deleted internl loop in pri-, whih is 7 nt wy from the : duplex if the rm is ounted (nd 6 nt wy if the rm is ounted). Only one mjor produt (~33 nt) ws produed in the reonstitution ssy (Fig. 4, lne 6, C- L). Further studies with the - or -ompromised mutnts (Supplementry Fig. 5) indited tht the 33-nt levge produt resulted from ortive proessing strting from BTLs to the mirna:mirna duplex. This result indited tht internl loop ws required for the nonil proessing of pri- nd proly served s the ounting site for proessing. To further define the length of the moleulr ruler of the pri-mirna miroproessor, we reted pri- mutnts y either nneling internl loop 3 (Fig. 4, lne 2, C- L3) or inserting three more nuleotides etween internl loops 2 nd 3 in the lower stem (Fig. 4, lne 8, C-ILS) p 7 p 2 p C- L2 C- L C- L3 C-ILS Insertion Figure 4 initites levge t 6 7 p from the ssrna-dsrna juntion in pri-mirnas. () In vitro reonstitution ssys with the -end-leled trnsripts of pri-f mutnt. Right, shemti illustrtion of the levge produts y. nd 2 indite two internl loops in the lower stems of pri-f. F-WT, wild-type pri-f. () In vitro reonstitution ssys with the -end-leled trnsripts of pri- deletion nd insertion mutnts in the lower stem. Right, shemti illustrtion of the levge produts y., 2 nd 3 indite three internl loops in the lower stems of pri-., wild-type pri-. () In vitro reonstitution ssys with the -end-leled trnsripts of pri- mutnts with fully omplementry lower stems. Right, shemti illustrtion of the levge produts y. C-LSA, pri- mutnts with the lower stem nneled. In, immunopreipittion, levge ssys nd RNA proessing were performed s desried in Figure 2. The positions of intt sustrtes, levge produts nd RNA mrkers re indited. Blk sterisks show the residul nonspeifi levge frequently present in the ssy system. Red sterisks on trnsripts indite the 32 P-leling positions. Lrge nd smll red rrowheds indite the predominnt nd minor levge sites, respetively, nd lrge nd smll turquoise rrowheds indite predominnt nd minor ortive proessing sites, respetively. In oth mutnts, the length of the predominnt levge produts remined the sme. These results indite tht the distne etween the referene nd proessing sites is ~6 7 p in plnts. The presene of internl loops nd ulges etween the referene nd proessing sites in the pri-mirnas might ffet the mesurement ury of the miroproessor in the pri-mirnas. To exmine this possiility, we reted nother set of pri- mutnts hroring ompletely liner lower stems of different lengths (Fig. 4). leved the pri-mirna mutnts similrly to the wy it leved wild-type pri- (Fig. 4,), further vlidting tht the proessing sites in pri-mirna were 6 7 p wy from the ssrna referene site. Together our in vitro results support the 5-nt model oserved in previous geneti nlyses nd more preisely pinpoint the levge sites of omplexes t 6 7 p wy from referene sites. djusts proessing sites lose to n internl loop Plnt pri-mirnas re highly heterogeneous in struture, nd mny of them lk lower stems with the optiml distne of 6 7 p etween referene ssrna regions nd mirna:mirna duplexes 25. We wondered whether plnt omplexes hve some flexiility in mesuring distnes nd/or n dditionl sensor system to identify proessing sites tht llow for the preise levge of pri-mirnas with struturl nd length heterogeneity. To filitte our experiments, we nneled internl loop 2 nd smll ulge etween internl loops 2 nd 3 in the lower stem of pri- (Fig. 5,, C-LS-7). Next we generted series of deletions or insertions sed on the C-LS-7 kone for levge ssys. In greement with the optiml length of nture struturl & moleulr iology dvne online pulition

6 3 Nture Ameri, In. All rights reserved. Figure 5 omplexes djust levge sites towrd the edge of internl loops through the helise domin. () In vitro reonstitution ssys with ATP. () Shemti illustrtion of the levge produts from. () In vitro reonstitution ssys with ( H) nd ATP. (d) Shemti illustrtion of the levge produts from. (e) In vitro reonstitution ssys without ATP. The shemti illustrtion of the levge produts from e ws similr to tht in d nd is thus omitted. In e, the sustrtes were the -end-leled trnsripts of pri- mutnts with vrying distnes etween internl loops nd the referene sites. Immunopreipittion, levge ssys nd RNA proessing were performed s desried in Figure 2. The positions of intt sustrtes, levge produts nd RNA mrkers re shown. Blk sterisks show nonspeifi levge produts frequently present in the ssy system. Lrge nd smll red rrowheds show the predominnt nd minor levge sites, respetively, nd lrge nd smll turquoise rrowheds indite the predominnt nd minor ortive proessing sites, respetively. Blk lines mrk the lotions of referene sites nd internl loops. Red sterisks on the trnsripts indite 32 P-leling positions. the moleulr ruler identified ove (Fig. 4), the levge effiieny of pri-mirnas ws highest when the mirna:mirna duplex ws 7 p wy from the referene internl loop (Fig. 5, lne 4, C-LS-7). Devition from this optiml distne ompromised the produtivity nd ury of the omplexes. Speifilly, if the distne ws shorter thn 6 p, inurtely ut the pri-mirnas t multiple lotions, resulting in pool of heterogeneous frgments (Fig. 5,). This result is onsistent with previous geneti studies showing tht the sequene nd struture of the lower stem (within ~5 nt wy from the mirna:mirna duplex) is ruil for the urte proessing of pri-mirna Notly, when the lower stem ws shorter thn the optiml length, we oserved more produts resulting from ortive proessing, wheres fewer produts were generted from produtive proessing (Fig. 5,). The inverse orreltion etween ortive nd produtive proessing on the sme pri-mirnas suggests steri hindrne for lunhing omplexes from two opposite diretions on the sme pri-mirna. Conversely, when the distne ws 2 p longer thn the optiml distne of 7 p, the predominnt levge sites remined t the sme lotion t the edge of n internl loop nd we oserved minor levge produts resulting from inurte levge (Fig. 5, lnes 6 nd 8, nd Fig. 5). This result suggests tht omplexes ould sense the loop nd djust their tlyti sites towrd the edge of the loop for levge. This levge pttern is reminisent of reent oservtion in niml Diers tht n sense loop regions to determine pre-mirna proessing 35. Consistent with this notion, numer of mirna:mirna duplexes were found to reside ner n internl loop tht is ~8 9 p wy from referene sites of pri-mirnas in genome-wide nlysis 28. Hene, omplexes n tolerte slight C-LS-2 C-LS-3 C-LS-4 C-LS-5 C-LS-6 C-LS-7 C-LS-8 C-LS-9 Lne H H H H H H H H Lne e C-LS-2 C-LS-4 C-LS-5 C-LS-6 C-LS-7 C-LS-8 C-LS-2 C-LS-4 C-LS-5 C-LS-6 C-LS-7 C-LS-8 C-LS-9 C-LS-9 Lne p C-LS-2 C-LS-3 C-LS-4 C-LS-5 C-LS-6 C-LS-7 C-LS-8 C-LS-9 d C-LS-2 C-LS-4 C-LS-5 C-LS-6 C-LS-7 C-LS-8 C-LS-9 in plnt 3 p 4 p 5 p 6 p 7 p 8 p 9 p 2 p 4 p 5 p 6 p 7 p 8 p 9 p devitions from the optiml 7-p distne etween the referene site nd the mirna:mirna duplex if the proessing position is lose to n internl loop. To further study how omplexes reognize the internl loop for proessing, we exmined possile role of the helise domin. Similrly to, with deletion of the helise domin ( H) leved pri-mirnas predominntly t sites 6 7 p wy from the ssrna referene sites regrdless of whether the proessing sites were t the edge of the internl loops (Fig. 5,d nd Supplementry Fig. 4). These results further vlidted the ide tht the nonil ruler of the miroproessor is 6 7 p in length. In ontrst, ( H) generted multiple levge produts when the internl loops devited 2 p from the optiml proessing sites, result suggesting tht pri-mirnas were inurtely proessed. This result is lso in line with previous study tht found tht deletion of the helise domin leds to inury of mirna iogenesis 32. The deletion of the entire helise domin potentilly hnges the overll struture of, resulting in ompromised proessing ury. To test this possiility, we repeted the levge ssys with wild-type in the sene of ATP nd GTP. In this senrio, the levge ptterns were essentilly identil to those with ( H) (Fig. 5e nd Supplementry Fig. 4d). Together these results indite tht the 6 dvne online pulition nture struturl & moleulr iology

7 3 Nture Ameri, In. All rights reserved. C-F Lne C- UL d C-T3 C-T2 Lne C-US mir66 mir66 C-F top mir66 mir66 6 p Internl loop C-T2+3 C-US5 C-US8 helise domin inspets n internl loop for proessing uthentiity, sitution reminisent of tht of fly Dier, whih reognizes single-strnd terminl-loop strutures of pre-mirnas through its helise domin 36. Struturl determinnts for pri-mirna ortive proessing Swpping the upper stem nd terminl region of pri- with their ounterprts from pri-f olished ortive proessing (Fig. 6), suggesting tht the lrge terminl loop might e disguised s ssrna-dsrna referene site, whih leds to the nonnonil reruitment of omplexes. To further pinpoint the strutures tht re required for ortive proessing, we reted series of muttions in the upper stem nd terminl loops of pri- (Fig. 6,). Deresing the size of the terminl loop either ompletely loked or sustntilly deresed ortive proessing (Fig. 6, lne 8, C-T2; lne 2, C-T4; lne 4, C-T5, nd Supplementry Fig. 5). Additionlly, terminl loop with less omplited top strutures lso llevited ortive proessing to some extent (Fig. 6, lne 0, C-T2+3). Conversely, the presene of n ~5- to 8-p linerized upper stem lose to lrge terminl loop fvored ortive proessing over produtive proessing (Fig. 6, lnes 6 nd 8). These in vitro results re onsistent with umultion in the trnsgeni plnts expressing these onstruts (Fig. f). Together, these dt suggest tht lrge terminl loop in pri-mirnas redily triggers nonnonil lunhing for miroproessor, whih results in ortive proessing. Similrly to produtive proessing, ll ortive proessing sites tended to our lose to internl loops (Figs. 5 nd 6). To vlidte C-US22 C-T4 C-T5 C- UL C-T3 C-T2 C-T2+3 C-T4 5 p 8 p C-T5 C-US5 Lne p Internl loop 5 p 7 p 22 p C-US8 Figure 6 Seondry struturl elements re required for ortive proessing in pri-. () In vitro reonstitution ssys with the -end-leled trnsripts of himeri pri- ontining the top region of pri-f. Right, shemti illustrtion of the levge produts y. () In vitro reonstitution ssys with the -end-leled trnsripts of pri- mutnts with vrious deletions, insertions nd se pirings in the upper stem nd terminl loop. () Shemti illustrtion of the produts of levge y from. (d) In vitro reonstitution ssys with the -end-leled trnsripts of pri- mutnt with full omplementry upper stem. Immunopreipittion, levge ssys nd RNA proessing were performed s desried Figure 2. The positions of intt sustrtes, levge produts nd RNA mrkers re shown. Blk sterisks show nonspeifi levge produts frequently present in the ssy system. Red sterisks on the trnsripts indite 32 P-leling positions. Blk lines show the distne etween the indited positions. Lrge nd smll red rrowheds show the predominnt nd minor expeted produtive levge sites, respetively, nd lrge nd smll turquoise rrowheds indite the predominnt nd minor ortive proessing sites, respetively. preisely the length of the moleulr ruler used in the ortive proessing, we first nneled smll internl loop (Fig. 6,, lne 4, C- UL) tht is p wy from the ortive proessing sites nd found tht the ortive proessing pttern remined unhnged. We lso reted pri- mutnt y inresing the length of the upper stem etween the internl loop nd the terminl loop (Fig. 6d, C-US22). reonstitution ssys with pri- US22 reveled tht the ortive proessing site ws 7 p wy from the lrge terminl loop. This result indites tht the moleulr ruler for ortive proessing is identil to tht used for produtive proessing (Figs. 4 nd 5). Mny other pri-mir-65s nd pri-s in Aridopsis lso hror omplited terminl strutures. To exmine whether ortive proessing ws pplied to these pri-mirnas, we tested pri-d in reonstitution ssys. We found tht pri-d, similrly to pri-, showed ortive proessing ptterns (Supplementry Fig. 6). These results further indite tht ortive proessing of pri-mirnas ounted for the low umultion of in the trnsgeni lines (Fig. ). Beuse pri-mir-65 nd pri- hve rnhed terminl loops ut yield higher in vivo levels of mir-65 nd expression, we wnted to lern how omplexes proess these pri-mirnas. In vitro ssys showed tht pri-mir-65, in ontrst to pri-, ws leved in predominntly produtive mnner (Supplementry Fig. 6). The preferene for produtive proessing over ortive proessing of pri-mir-65 is proly due to its reltively smller terminl loop size nd the shorter distne etween the terminl loop nd the mirna:mirna duplex. Notly, pri- ould never e ut y in our reonstitution system (H.Z., C.G. nd X.Z., unpulished dt), result suggesting tht there might e n lterntive pthwy ypssing nonil -medited proessing in vivo. nture struturl & moleulr iology dvne online pulition 7

8 3 Nture Ameri, In. All rights reserved. Figure 7 Bidiretionl proessing of pri-mirnas (BTLs) extends eyond the pri- fmily in Aridopsis. () Exmples reovered from nlyses of degrdome nd srna lirries. () Exmples reovered from nlysis of srna lirries only. In nd, mirna nd mrna sequenes re shown in turquoise nd red, respetively. Red rrowheds nd numers show produtive proessing sites nd ounts of mirna reds. Blue rrowheds nd numers show positions nd ounts of srnas resulting from pprent ortive proessing events. Pink rrowheds nd numers show positions nd ounts of srnas resulting from unknown proessing events. Gold rrowheds nd numers show positions nd ounts of degrdome reds 22,4 43. The distnes from the ortive proessing sites to terminl loops or etween proessing sites re etween the two lk lines. Reds of mirnas re not shown euse they re not unique for individul pri-mirna prlogs. Minor forms of mirna: mirna duplexes tht re progressively frther wy from predominnt mirna:mirna loi re shown in Supplementry Tles 2 nd 3. () In vitro reonstitution ssys with syntheti TLBed pri-mirna. Right, shemti illustrtion of the levge produts y. Protein immunopreipittion, levge ssys nd RNA proessing were performed s desried in Figure 2. The positions of intt sustrtes, levge produts nd RNA mrkers re shown. Red sterisks on trnsripts indite the 32 P-leling positions. Turquoise nd purple rrowheds indite ortive nd produtive proessing sites, respetively. Lrge nd smll rrowheds indite predominnt nd minor levge sites, respetively. Bidiretionl proessing extends eyond pri-s We found tht 77 out of 232 Aridopsis hirpins from mir hd BTLs. To exmine whether idiretionl proessing of pri-mirnas ours eyond the pri- fmily, we first explored eight previously desried degrdome dt sets 37 for evidene of ortive proessing. Degrdome sequening determines the RNAs tht hve monophosphtes, whih inlude remnnts of -tlyzed pri-mirna proessing 27, mong other types of proessed RNAs. We defined ortive proessing y the presene of degrdomeinferred remnnts ending etween 5 nd 7 nt from the loopproximl end of the mirna-ontining helix. Of the 55 hirpins with norml, unrnhed loops, 6 hd evidene of ortive proessing (3.9%). Seven of the 77 (9.%) hirpins with BTLs hd suh evidene (Fig. 7 nd Supplementry Fig. 7). Produts derived from ortive proessing of pri-mirnas, exemplified y pri-, re proly unstle, nd therefore mny my espe detetion y degrdome sequening. We rgue tht if intermedite proessing frgments from ortive proessing re nerly self omplementry, progressive levge y omplexes might generte some smll RNA (srna) remnnts other thn mirna:mirna duplexes. We next mined high-throughput srna sequening dt sets 4 to identify ny srna yproduts derived from potentil ortive proessing. We indeed oserved mny srnas tht were inditive of idiretionl proessing (Fig. 7 nd Supplementry Fig. 7). Some of these pri-mirnas were identil to those reovered from the degrdome-inferred pproh desried ove (Fig. 7). In one instrutive se, we found tht pri-mir-825 hrors rnhed loops in its lower stem nd norml loop in the upper stem. Notly, the diretions of produtive nd ortive proessing re lso swithed for pri-mir-825. Similr studies in rie lso reovered severl pri-mirnas, inluding pri-, result suggesting tht the presene of idiretionl proessing of the sme pri-mirnas extends eyond Aridopsis (Supplementry Fig. 7). pri-mir-7 pri-mir-394 pri-mir p 2 Syntheti 8 pri-mirna ATP p 3 7 p p pri-mir-447 pri-mir p pri-mir-393 pri-mir-859 pri-mir p To further test whether there is generl predisposition towrd idiretionl proessing for TLBed pri-mirnas, we engineered n rtifiil miniture TLBed pri-mirna (Fig. 7). In vitro reonstitution ssy with this syntheti pri-mirna indeed reveled the onurrent presene of oth produtive nd ortive proessing. This result further suggests tht idiretionl proessing of pri-mirnas with lrge BTLs might e ommon senrio in plnts. The terminl loop influenes the stility of pre-mirnas The presene of ortive proessing of pri- ws not solely responsile for the lowest umultion in vivo euse hlf of the pri- ws still proessed through produtive proessing (Fig. 2). To etter understnd whether the TLBed pre-mirnas were proessed similrly to their pri-mirnas, we repeted the in vitro reonstitution ssys with -end-leled pre- nd pre-f. We oserved only one levge frgment (2 nt) in the proessing of pre- nd pre-f (Fig. 8), result inditing tht omplexes urtely proessed the pre-mirnas nd lrge terminl loop did not trigger ortive proessing for pre-. We then ompred the stedy-stte undnes of pre-mirnas tht ontin different terminl strutures. RNA lot nlysis of totl RNAs prepred from stle trnsgeni plnts or the N. enthmin trnsient system showed tht pre-mirnas with smll terminl loops (MIR66 T nd T2) were esier to detet thn those with lrge terminl loops (MIR66 top nd MIR66 T3) (Fig. 8 nd Supplementry Fig. 8). In ddition, lrge internl loop in the upper stem or multirnhed terminl strutures lso ffeted pre-mirna stility (Fig. 8 nd Supplementry Fig. 8, MIR66 T2+3 nd MIR66 Ts3). These results indite tht the seondry strutures of terminl regions in pre-mirnas regulte their stility, further ontrolling homeostsis of mirna umultion in vivo p 9 6 p p p p p p 6 p p dvne online pulition nture struturl & moleulr iology

9 3 2 r t i l e s 3 Nture Ameri, In. All rights reserved. Figure 8 The terminl loop ffets the stedystte undne ut not the proessing pttern of pre-mirnas. () In vitro reonstitution ssys with the -end-leled trnsripts with pre- nd pre-f. Protein immunopreipittion, levge ssys nd proessing of RNA produts were performed s desried in Figure 2. The positions of intt sustrtes, levge produts nd RNA mrkers re shown. Right, shemti illustrtion of the levge produts y. Red sterisks on the trnsripts indite 32 P-leling positions. Blk rrows show the expeted proessing sites. () RNA lot nlysis of pre- in the stle trnsformnts expressing 35S-MIR66 mutnts. The RNA lot ws proed using 32 P-leled oligonuleotide proes omplementry to juntion regions of nd the upper stem in pre-. Blk sterisks indite pre-mirnas. mir-64 ws proed s loding ontrol. () A model for idiretionl proessing of pri-mirna y. BTLs regulte mirna iogenesis y triggering ortive proessing of pri-mirnas nd destilizing pre-mirnas. Exo- nd endo-rnses re shown in yellow. DISCUSSION A nonil pthwy for pri-mirna proessing is tht RNse III proteins nd their prt- ners define their initil ut t position wy from the ssrna-dsrna juntion in the lower stems to relese pre-mirnas 2. A nonnonil pthwy for mirna iogenesis lso exists ~2 nt for some plnt pri-mirnas tht hror long liner stem-loop struture (pri-mir-59 nd pri-mir39): initites sequentil uts lose to the terminl loop rther thn t the se until the relese of mirna:mirna duplexes 28. Here we propose new mode of tion for pri-mirna proessing, s protein proesses the sme pri-mirnas in two diretions, either from the lower stem to the terminl loop or vie vers (Fig. 8). This senrio ours s result of the struturl heterogeneity of plnt pri-mirnas. Mny pri-mirnas, inluding pri-, ontin lrge terminl loops, whih my disguise ssrna-dsrna juntion to reruit omplexes for levge. This outside-in mehnism hs two outomes: if goes from se to loop, it produes mture mirnas; if it goes in the opposite diretion, it destroys them. Given tht pri-mirnas ould e proessed forwrds or kwrds in plnts, the question rises of how omplexes proess primirnas with lrge terminl loops. Our results hve shown tht the inding orienttion of omplexes in pri-mirnas is key ftor in determining whether produtive or ortive proessing events our. Although extly how omplexes ssoite with primirnas or vie vers is unler in plnts, some struturl elements in pri-mirna my ontriute to the preferene of ssoition (Fig. 8). First, lrge terminl loops nd long upper stems promote nonnonil proessing (Fig. 6 ). Seond, the presene of ssrna region in the lower stem is required for nonil funtion euse the deletion of this segment ompletely suppresses produtive proessing (Fig. 4). Third, the presene of n internl loop in the lower stem proly suppresses produtive proessing nd filittes ortive proessing (Fig. 5). Similr results hve lso een 0 Lne pre- pre-f 2 nt mirna mirna pre- pre-f Empty vetor ~7 p ~7 p Helise Helise Helise ~2 nt Helise Helise MIR66 top reported in niml systems 2, result suggesting tht in oth plnts nd nimls, internl loop strutures in the stem influene the rtio of produtive nd ortive proesses 2. Notly, in our study, Mfold nlysis 4 suggested the presene of lrge internl loops in the lower stems of pri- nd pri-. These pri-mirnas were effiiently proessed in vivo ut not in vitro. It is tempting to propose tht the seondry strutures of these pri-mirnas might e different from the predited strutures. Alterntively, these pri-mirnas my hve unppreited proessing mehnisms tht ypss nonil pri-mirna proessing. We report numerous TLBed pri-mirnas in Aridopsis nd dditionl ses in rie tht re inditive of idiretionl proessing. Suh proessing method my lso e present ut not esily deteted in mny other TLBed pri-mirnas for exmple, pri- euse of extremely low expression of pri-mirnas or instility of ortive proessing produts. Conversely, we hve no reson to exlude the presene of idiretionl proessing in pri-mirnas with liner-hirpin strutures ut lso lrge internl or terminl loops, s oserved in in vitro ssys (Fig. 6) nd in plnt (Supplementry Fig. 7). It hs een suggested tht omplexes initilly proess pri-mrnas t distne of ~5 nt from either ssrna-dsrna juntion or n internl unstrutured region Here we provide MIR66 T MIR66 T2 MIR66 T3 Helise ~6 p MIR66 T2+3 MIR66 T s3 pre-mirna mir-64 levge First Seond nture struturl & moleulr iology dvne online pulition

10 3 Nture Ameri, In. All rights reserved. diret iohemil evidene to oost this model nd more preisely pinpoint the levge sites. We lso demonstrte tht the nonil ruler system is pplile to oth produtive nd ortive proessing (Fig. 8). The referene site for miroproessor reognition n e lrge internl or terminl loop nd is not neessrily ounted from the ssrna segments t the lower stem of pri-mirnas. Genome-wide nlysis hs shown tht hlf of the pri-mirnas, whih re proly sujeted to nonil mirna proessing, hror n unpired region or loop 7 nt wy from the loop-distl levge sites in the lower stem 25. However, the miroproessor in plnts lso hs ertin degree of plstiity nd llows for produtive proessing from strutures tht re slightly different from the optiml 6 7 p, espeilly in senrios in whih the proessing sites re ner n internl loop. We propose tht my use nonnonil ruler system to sense the loop region nd djust the levge sites in pri-mirnas, thus permitting more fitness in generting uniform mirnas (Fig. 5). The sensing of the internl loop is pprently through the helise domin of (Fig. 5). However, omplexes do not fvor distnes shorter thn 6 p from the ssrna-dsrna referene site to the potentil proessing position. Our iohemil results re onsistent with the previous omputtionl disovery tht Aridopsis pri-mirnas hve evolved to ontin unpired regions or loops 2 4 nt wy from the levge sites only rrely 25. Tken together our results provide new insight into understnding the omplited proessing mehnism of pri-mirnas in plnts nd eyond. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk H. Koiw for pdonrzeo-hyl (Deprtment of Hortiulture, Texs A&M University) nd D. Shippen, G. Kpler, F. Qio, P.W. Li nd M. Klein for stimulting disussions nd ritil review of the mnusript. We lso thnk F. Hu, H. Xu, Z. Zhng, C. Hung nd C. Lu for tehnil ssistne. The work ws supported y grnts from the US Ntionl Siene Foundtion (NSF) (MCB-095) nd NSF CAREER (MCB ), the US Ntionl Institutes of Helth (R2AI0975) nd the Welh foundtion (A-777) to X.Z. A.L. ws supported y NSF-REU (MCB-23287). Y.Z. ws supported y the Chinese Sholrship Counil. AUTHOR CONTRIBUTIONS H.Z., C.C.-G. nd X.Z. designed experiments. Y.Z. rried out geneti reserh. H.Z. nd C.C.-G. performed iohemil studies. M.J.A. onduted degrdome nlysis. Y.-T.Z. nd X.-J.W. worked on srna dt set nlysis. A.L., C.G., L.D. nd Z.L. prtiipted in experiments or provided mterils nd intelletul input for the work. X.Z. nd H.Z. wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html.. Kim, V.N., Hn, J. & Siomi, M.C. Biogenesis of smll RNAs in nimls. Nt. Rev. Mol. Cell Biol. 0, (09). 2. Hn, J. et l. Moleulr sis for the reognition of primry mirornas y the Drosh-DGCR8 omplex. Cell 25, (06). 3. Brtel, D.P. MiroRNAs: genomis, iogenesis, mehnism, nd funtion. Cell 6, (04). 4. Liu, J. et l. Argonute2 is the tlyti engine of mmmlin RNAi. Siene 5, (04). 5. Voinnet, O. Origin, iogenesis nd tivity of plnt mirornas. Cell 36, (09). 6. Guo, H., Ingoli, N.T., Weissmn, J.S. & Brtel, D.P. Mmmlin mirornas predominntly t to derese trget mrna levels. Nture 466, (0). 7. Czeh, B. & Hnnon, G.J. Smll RNA sorting: mthmking for Argonutes. Nt. Rev. Genet. 2, 9 3 (). 8. Bzzini, A.A., Lee, M. & Girldez, A. Riosome profiling shows tht mir-4 redues trnsltion efore using mrna dey in zerfish. Siene 336, (2). 9. Djurnovi, S., Nhvi, A. & Green, R. mirna-medited gene silening y trnsltionl repression followed y mrna dedenyltion nd dey. Siene 336, (2). 0. Li, S. et l. MiroRNAs inhiit the trnsltion of trget mrnas on the endoplsmi retiulum in Aridopsis. Cell 53, (3).. Chen, X. Plnt mirornas t glne. Semin. Cell Dev. Biol. 2, 78 (0). 2. Ren, G. et l. Regultion of mirna undne y RNA inding protein TOUGH in Aridopsis. Pro. Ntl. Ad. Si. USA 09, (2). 3. Mhid, S., Chen, H. & Yun, A. Moleulr insights into mirna proessing y Aridopsis thlin SERRATE. Nulei Aids Res. 39, (). 4. Yng, S.W. et l. Struture of Aridopsis HYPONASTIC LEAVES nd its moleulr implitions for mirna proessing. Struture 8, (0). 5. Dong, Z., Hn, M. & Fedoroff, N. The RNA-inding proteins HYL nd SE promote urte in vitro proessing of pri-mirna y. Pro. Ntl. Ad. Si. USA 05, (08). 6. Grigg, S.P., Cnles, C., Hy, A. & Tsintis, M. SERRATE oordintes shoot meristem funtion nd lef xil ptterning in Aridopsis. Nture 437, (05). 7. Hn, M.H., Goud, S., Song, L. & Fedoroff, N. The Aridopsis doule-strnded RNA inding protein HYL plys role in mirorna-medited gene regultion. Pro. Ntl. Ad. Si. USA 0, (04). 8. Kurihr, Y., Tkshi, Y. & Wtne, Y. The intertion etween nd HYL is importnt for effiient nd preise proessing of pri-mirna in plnt mirorna iogenesis. RNA 2, 6 22 (06). 9. Loes, D., Rllplli, G., Shmidt, D., Mrtin, C. & Clrke, J. SERRATE: new plyer on the plnt mirorna sene. EMBO Rep. 7, (06).. Vzquez, F., Gsiolli, V., Crete, P. & Vuheret, H. The nuler dsrna inding protein HYL is required for mirorna umultion nd plnt development, ut not posttrnsriptionl trnsgene silening. Curr. Biol. 4, (04). 2. Hirsh, J. et l. Chrteriztion of 43 non protein-oding mrna genes in Aridopsis, inluding the MIR62-derived trnsripts. Plnt Physiol., 92 4 (06). 22. Xie, Z. et l. Expression of Aridopsis MIRNA genes. Plnt Physiol. 38, (05). 23. Cuperus, J.T. et l. Identifition of MIR390 preursor proessing-defetive mutnts in Aridopsis y diret genome sequening. Pro. Ntl. Ad. Si. USA 07, (0). 24. Mteos, J.L., Bologn, N.G., Chorosteki, U. & Pltnik, J.F. Identifition of mirorna proessing determinnts y rndom mutgenesis of Aridopsis MIR72 preursor. Curr. Biol., (0). 25. Song, L., Axtell, M. & Fedoroff, N. RNA seondry struturl determinnts of mirna preursor proessing in Aridopsis. Curr. Biol., 37 4 (0). 26. Werner, S., Wollmnn, H., Shneeerger, K. & Weigel, D. Struture determinnts for urte proessing of mir72 in Aridopsis thlin. Curr. Biol., (0). 27. Addo-Quye, C. et l. Slied mirorna trgets nd preise loop-first proessing of MIR39 hirpins reveled y nlysis of the Physomitrell ptens degrdome. RNA 5, (09). 28. Bologn, N.G., Mteos, J.L., Bresso, E.G. & Pltnik, J.F. A loop to se proessing mehnism underlies the iogenesis of plnt mirornas mir39 nd mir59. EMBO J. 28, (09). 29. Zhu, H. et l. Aridopsis Argonute0 speifilly sequesters mir66/65 to regulte shoot pil meristem development. Cell 45, ().. Zhng, Z. & Zhng, X. Argonutes ompete for mir65/66 to regulte shoot pil meristem development. Curr. Opin. Plnt Biol. 5, (2). 3. Zuker, M. Mfold we server for nulei id folding nd hyridiztion predition. Nulei Aids Res. 3, (03). 32. Liu, C., Axtell, M.J. & Fedoroff, N.V. The helise nd RNse III domins of Aridopsis Dier-like modulte tlyti prmeters during mirorna iogenesis. Plnt Physiol. 59, (2). 33. Mnvell, P.A. et l. Fst-forwrd genetis identifies plnt CPL phosphtses s regultors of mirna proessing ftor HYL. Cell 5, (2). 34. Weinerg, D.E., Kotro, N.K., Dinshw J. Ptel, D. & Brtel, D. The inside-out mehnism of Diers from udding yests. Cell 46, (). 35. Gu, S. et l. The loop position of shrnas nd pre-mirnas is ritil for the ury of Dier proessing in vivo. Cell 5, (2). 36. Tsutsumi, A., Kwmt, T., Izumi, N., Seitz, H. & Tomri, Y. Reognition of the pre-mirna struture y Drosophil Dier-. Nt. Strut. Mol. Biol. 8, (). 37. Addo-Quye, C., Eshoo, T.W., Brtel, D.P. & Axtell, M.J. Endogenous sirna nd mirna trgets identified y sequening of the Aridopsis degrdome. Curr. Biol. 8, (08). 38. Germn, M.A. et l. Glol identifition of mirorna-trget RNA pirs y prllel nlysis of RNA ends. Nt. Biotehnol. 26, (08). 39. Gregory, B.D. et l. A link etween RNA metolism nd silening ffeting Aridopsis development. Dev. Cell 4, (08).. M, Z., Coruh, C. & Axtell, M.J. Aridopsis lyrt smll RNAs: trnsient mirna nd smll interfering RNA loi within the Aridopsis genus. Plnt Cell 22, (0). 4. Brrett, T. et l. NCBI GEO: rhive for funtionl genomis dt sets updte. Nulei Aids Res. 4, D99 D995 (3). 42. Zhng, X., Henriques, R., Lin, S.S., Niu, Q. & Chu, N.H. Agroterium-medited trnsformtion of Aridopsis thlin using the florl-dip method. Nt. Proto., (06). 43. Zhng, X. et l. Cuumer mosi virus enoded 2 suppressor inhiits Aridopsis Argonute levge tivity to ounter plnt defense. Genes Dev., (06). 0 dvne online pulition nture struturl & moleulr iology

11 3 Nture Ameri, In. All rights reserved. ONLINE METHODS Vetor onstrution. The mjority of onstruts were mde using Gtewy system (Invitrogen) 42,43. The primers for the onstrution of ll vetors re listed in Supplementry Tle. Genes enoding mirnas, inluding the onise nd extended forms, were loned from Aridopsis genomi DNA. These genes were introdued into pentr/d vetors, onfirmed y sequening nd trnsferred into the pba-dc destintion vetors using LR Clonse (Invitrogen). Chimeri primry mirnas with extended forms were onstruted s desried elow. For pentr pri-f B+T (extended form, where B is the se nd T is the top), we repled the se nd top sequenes of pri-f with the ounterprts from pri-, with the f duplex kept intt. Briefly, the extended form of pentr pri- ws used s templte for two sequentil mutgenesis PCRs using two sets of primers: -to-f forwrd nd -to-f reverse for the first PCR (PCR) nd -to-f forwrd nd -to-f reverse for the seond PCR (PCR2). Then the mutted pentr pri- (extended form) ws further used s templte for overlpping PCR using two sets of primers (ftl forwrd nd M3 reverse, nd M3 forwrd nd fls reverse). The resultnt PCR frgments were mixed, mplified using forwrd nd reverse primers, nd further loned into pentr/d to generte pentr pri-f B+T (extended form). For pentr pri- f B+T (extended form), we exhnged the se nd top sequenes of pri- with the ounterprts from pri-f using strtegy similr to tht used for pentr pri-f B+T (extended form) desried ove. The primers for sequentil mutgenesis PCRs were f-to- forwrd nd f-to- reverse, nd f-to- forwrd nd f-to- reverse. The primers for overlpping PCRs were ftl forwrd nd M3 reverse, M3 forwrd nd fls reverse, nd f forwrd nd reverse. For pentr/d pri-f T (extended form), we exhnged the top region of f with tht from. We used pentr pri-f (extended form) s templte for three rounds of extension PCRs. The three groups of primers were TL-f forwrd nd M3 reverse, TL-f forwrd 2 nd M3 reverse, nd TL-f forwrd 3 nd M3 reverse. The resultnt PCR frgment ws mixed with nother PCR frgment generted with M3 forwrd nd TL-f reverse primers using pentr pri-f (extended form) s templte for overlpping PCR. The overlpping PCR produt ws mplified using f forwrd nd f reverse primers nd loned into pentr/d to generte pentr pri-f T (extended form). For pentr/d pri- f T (extended form), we exhnged the top region of with tht from f using strtegy similr to the one for the himeri vetor pentr pri-f T (extended form) desried ove. The two sets of primers for extension PCRs were f TL- forwrd nd M3 reverse, nd f TL- forwrd 2 nd M3 reverse. The primers for the overlpping PCRs were M3 forwrd nd f TL- reverse, nd forwrd nd reverse. The finl PCR produt ws loned into pentr/d to generte pentr f T (extended form). For pentr/d pri-f B (extended form), we exhnged the se region of f with tht of using strtegy similr to tht used with the himeri plsmids desried ove. The two sets of primers for the extension PCRs were f TL- forwrd nd M3 reverse, nd f TL- forwrd 2 nd M3 reverse. The primers for the overlpping PCRs were M3 forwrd nd f TL- reverse, nd forwrd nd reverse. The resultnt PCR produt ws loned into pentr/d vetor. This pentr vetor ws used s templte for n dditionl round mutgenesis PCR using the primers LS GT-to-g forwrd nd Ls GT-to-g reverse to rete pentr pri-f B (extended form). For pentr/d pri- f B (extended form), we repled the se region of with tht of f in the kone. The extension PCRs were onduted with three sets of primers (TL-f forwrd nd M3 reverse, TL-f forwrd 2 nd M3 reverse, nd TL-f forwrd 3 nd M3 reverse). Overlpping PCR ws then performed using the primers M3 forwrd nd TL- reverse, nd f forwrd nd f reverse. The resultnt PCR produt ws loned into pentr/d. This pentr vetor ws used s templte for n dditionl round of mutgenesis PCR using the primers fls -to-gt forwrd nd fls -to-gt reverse to rete pentr pri- f B (extended form). For ll onise forms of the pentr pri- nd pri-f vetors, the short PCR produts were generted using the wild-type nd himeri mutnts of pentr pri- nd pri-f (extended form) s templtes nd the primers of forwrd nd reverse, nd f 48 forwrd nd f 48 reverse. The resultnt frgments were loned into pentr/d to rete the onise wild-type nd mutnt forms of pentr pri- nd pri-f. pentr pri- L2, pri- ILS, pri-f L, pri-mir- 66 UL, pri- IUS nd pri- US-5 were reted through mutgenesis PCRs using pentr pri- (onise form) s kone nd the orresponding primers listed in Supplementry Tle. pentr pri- US-8 nd pri- US-22 were reted through mutgenesis PCRs using pentr pri- US-5 s kone nd the orresponding primers listed in Supplementry Tle. LR retions yield ~-nt single-strnded segments flnking either the or end of the lower stems of pri-mirnas nd might lter the folding ptterns of pri-mirnas. To void this potentil issue, we reted series of onise pri-mirnas using lssi restrition digestion nd ligtion. Briefly, PCR produts were mplified using pentr pri- (onise form) nd pentr pri-f (onise form) s templtes nd the orresponding primers listed in Supplementry Tle. The purified PCR frgments were ut with XI nd SI. The resultnt frgments were ligted into XI- nd SI-treted pba002 to produe pba002 pri-, pba002 pri-f, pba002 pri- B nd pba002 pri-f B. For pba002 pri- T, we nneled two long oligonuleotides, C88- nd C88-2. PCR ws performed using the nneled oligonuleotides s templte nd primers ( XI forwrd nd SI reverse) tht were self mplified. The resultnt PCR produts were ut with SI nd XI nd ligted to the sme enzyme-treted pba002. pba002 pri-f T, pri- T2+3, pri- T3, pri T2, pri- T, pri- T4, pri- T5 nd pri- Ts3 were reted using strtegy similr to tht used with pba002 pri- T desried ove with the orresponding primers listed in Supplementry Tle. For pba002 pri- L3, pentr pri- (onise form) ws used s templte for two sequentil mutgenesis PCRs using the orresponding primers listed in Supplementry Tle. The resultnt mutted pentr pri- ws further used s templte for the PCR retion with two primers, XI forwrd nd SI reverse. The finl PCR produt ws ut with XI nd SI nd suloned into pba002. For pentr pri- LSA-5, pba002 pri- L3 ws used s templte for one mutgenesis PCR using the orresponding primers listed in Supplementry Tle. The resultnt frgments were loned into pentr/d vetors. pba-6my-hyl ws generted y the LR retion etween pdonrzeo- HYL nd pba-6my-dc. SE ws mplified from Aridopsis DNA with the two primers listed in Supplementry Tle nd loned into pentr vetor to generte pentr-se. The SE gene ws further trnsferred into pba-dc-3ha or pba-flg-4my-dc to yield pba-se-3ha or pba-flg-4my-se through n LR retion. For pba-2flg-4my-, ws first mplified from pta02- Flg- (ref. 8) nd suloned into pba002 to rete pba002-2flg- 4My-. All pba002-2flg-4my- mutnts, inluding (E7Q), (E696Q), (E7Q E696Q) nd ( H), were generted y mutgenesis PCR nd overlpping PCR using the primers listed in Supplementry Tle. Plnt mteril nd growth onditions. The plnt-growing onditions were previously desried 44. Wild-type A. thlin plnts (Col-0 nd Ler) were trnsformed with the reomintion inry vetors ontining the onise forms of pri-mirnas nd n empty vetor y the florl dip trnsformtion method. Seeds from infiltrted plnts were seleted on stndrd Murshige nd Skoog (MS) medium ontining the pproprite seletive gents: 0 mg l glufosinte mmonium (Sigm) nd mg l reniillin (Sigm). RNA lot nlyses. Totl RNA ws extrted using TRIzol regent from Agroterium-trnsfeted N. enthmin leves or Aridopsis tissues, inluding 7- or 0-dy-old seedlings, depending on the desired ssys. Northern lots were performed s desried 2,3,29. Eh lne ontined 0 µg of totl RNA. Blots were hyridized with 32 P-rdioleled oligonuleotide proes omplementry doi:0.038/nsm.2646 nture struturl & moleulr iology

12 3 Nture Ameri, In. All rights reserved. to mir-65 or. U6 served s the loding ontrol in the trnsient ssy. mir-64 served s loding ontrol in lots from trnsgeni plnts. RNA lots were deteted fter exposure to phosphor plte nd quntified using Quntity One Version ording to the mnufturer s instrutions (Bio-Rd). Mteril preprtion for trnsient nd in vitro proessing experiments. For trnsient experiments, 4-week-old N. enthmin leves were infiltrted with n Agroterium tumefiens strin, ABI, hroring vriety of inry plsmids. The optil density t 0 nm (OD 0 ) of the A. tumefiens ultures ws diluted to 0.4 in most of the experiments. For the in vitro experiment proessing omplexes (wild type or mutnt), we infiltrted too with three ABI strins hroring (or mutnt), HYL nd SE together. The OD 0 of the pba002-2flg-4my- (or mutnts) ulture ws 0.8. The OD 0 vlues of pba-6my-hyl, pba-se-3ha nd pba-flg-4my-se were diluted to 0.. The trnsfeted leves were olleted 2 d fter groinfiltrtion. In vitro trnsription of RNA. RNA sustrtes were trnsried under the T7 promoter in vitro using PCR-generted templtes. The templtes nd primers used for PCRs nd the syntheti pri-mirnas re listed in Supplementry Tle. The in vitro trnsription of RNAs ws rried out in one -µl retion inuted for 3 h or overnight t 37 C s follows: µl of DNA templte ( ng), 4 µl of 5 trnsription uffer (0 mm HEPES, ph 7.5, 0 mm spermidine, 0 mm DTT, 25 mm MgCl 2 nd mm of eh NTP), µl of RNse inhiitor (Amion), 2 µl of T7 RNA polymerse nd 2 µl of wter. DNse-treted RNA ws frtionted y 6% polyrylmide nd 8 M ure gel (denturing gel), eluted overnight from gel slies in RNA elution uffer (0.3 M NA, ph 5.5, nd 2% SDS) using Thermomixer R (Eppendorf) t 42 C nd,0 r.p.m., preipitted with ethnol nd stored in diethylpyroronte (DEPC)-treted wter. Leling of RNA. For internl leling, [α- 32 P]-UTP (PerkinElmer) ws inluded in the NTP mixture ( mm CTP, mm ATP, mm GTP nd 4 mm UTP) for the in vitro trnsription desried ove. For -end leling, nonrdiotive purified pri- or pre-mirnas (5 pmol) were dephosphorylted with lkline phosphtse (New Englnd Biols) nd leled with [γ- 32 P]-ATP (PerkinElmer) using T4 polynuleotide kinse (New Englnd Biols) ording to the mnufturer s protool. For -end leling, nonrdiotive purified pri-mirna ws ligted with [α- 32 P]-pCp (PerkinElmer) using T4 RNA Ligse (New Englnd Biols) ording to the mnufturer s instrutions. All the leled RNAs were frtionted with 6% denturing gel, eluted from gel slies in RNA elution uffer, ethnol preipitted with GlyoBlue (Amion) nd stored in uffer ( mm KCl nd mm Tris-HCl, ph 7.5). Leled RNAs were folded y heting to 95 C for 2 min, slowly ooled down to room temperture nd normlized to ~2 0 3.p.m. µl. Immunopreipittion nd in vitro reonstitution ssy. Immunopreipittion ws performed s desried 29 with some modifitions. Too smples were ground in liquid nitrogen, nd protein srna omplexes were extrted using five volumes of immunopreipittion uffer ( mm Tris-HCl, ph 7.5, 0 mm KCl, 5 mm MgCl 2, 5 mm DTT, 0.2 mm EDTA, ph 8.0, 0.2% Triton X-, mm PMSF, 2% glyerol nd 0.3% (vol/vol) proteinse inhiitor oktil (Sigm)) nd tlet per 25 ml immunopreipittion uffer of EDTAfree protese inhiitor oktil (Rohe). After removl of insolule mterils y entrifugtion twie t 6,000g for 5 min t 4 C, extrts were inuted with nti-flg M2 mgneti eds (Sigm, M8823) for 2 h t 4 C. The eds were wshed three times with the immunopreipittion uffer, nd tgged omplexes were eluted twie y inution with the immunopreipittion uffer ontining µg ml 3 Flg peptide (Sigm) for min t 4 C. The eluted frtion ws inuted with nti-my grose eds (Sigm, A74) for.5 h. The eds were wshed three times with immunopreipittion uffer nd then three times with wshing uffer ( mm Tris-HCl, ph 7.5, mm DTT, 4 mm MgCl2 nd mm KCl). Briefly, in vitro levge ssys were performed in totl volume of µl in mm Tris-HCl, ph 7.5, 53 mm KCl, 4 mm MgCl 2, mm DTT, 7.5 mm ATP, mm GTP nd u µl RNse Inhiitor (Amion), inluding µl of leled RNA (~2 0 3.p.m.) nd 5 µl of the immunopurified proteins in wshing uffer. The retion mixture ws inuted t 37 C for 0 90 min. For pre-mirna, the retion ws inuted for 0 5 min. After extrtion with phenol-hloroform nd ethnol preipittion with GlyoBlue (Amion), the proessing produts were frtionted using 5% denturing gels. The RNA mrker used ws Dede mrker (Amion), whih ws -end leled ording to the mnufturer s protool with [γ- 32 P]-ATP (PerkinElmer). The denturing gel ws fixed in B uffer (% ethnol, 0% ette id nd 5% glyerol) nd dried with Gel Dryer (Bio-Rd) under the grdient yle t C for 5 h. The proessed RNA produts were deteted fter exposure overnight to phosphor plte nd quntified with Quntity One Version ording to the mnufturer s instrutions (Bio-Rd). The originl imges of the key lots nd utordiogrphs used in this study re shown in Supplementry Figure 9. Western lot nlysis. Assys of western lots were performed s desried 29. The ntiodies for immunopreipittion were nti-flg M2 mgneti eds (Sigm, M8823) nd nti My grose eds (Sigm, A74). Antiodies for western lot nlysis were nti-flg (Sigm, F365, :5,000 dilution), nti-my (Sigm, C395, :8,000 dilution) nd nti-ha (Snt Cruz, s-7392, :,000 dilution). Seondry ntiodies were sheep-developed nti-mouse IgG (GE, NA93V, :0,000 dilution) nd donkey-developed nti-rit IgG (GE, NA934V, :0,000 dilution). RNA seondry struture. The seondry strutures were predited from the Mfold we server ( Semiquntittive PCR. Totl RNAs were prepred from ontrol plnts or different trnsformnts nd treted with DNse efore eing sujeted to DNA synthesis using Supersript III reverse trnsriptse (Invitrogen) primed y oligo(dt). The primry mirna genes were mplified with the primers listed in Supplementry Tle. The elongtion ftor- (EF-) gene ws inluded s n internl ontrol 29. RNA ligse-medited RACE. RNA ws isolted from the wild-type strin Col-0 nd pooled seedlings of T trnsgeni plnts expressing 35S-MIR66. One-hundred piomoles of dptor (rgrururcrargrargrurur CrUrArCrArGrUrCrCr GrArCrGrArUrC) ws diretly ligted with 5 µg of totl RNA. After ligtion, first-strnd DNAs were synthesized using Supersript III Reverse Trnsriptse (Invitrogen, CA) nd pri- speifi primer ( RACE outer reverse). The DNA ws treted with RNse H to remove the RNA strnd nd mplified for two rounds using two sets of primers ( RACE outer forwrd nd RACE outer reverse, nd RACE inner forwrd nd RACE inner reverse). The distint PCR produts were loned into the pentr vetor (Invitrogen, CA) nd sequened using either M3 forwrd or M3 reverse primers. Computtionl nlysis of degrdome lirries nd srna dtses. Sequenes of ll Aridopsis mir hirpins (mir 9) were retrieved from genomi oordintes with n extension of nt on either side. Degrdome dt sets 37 were downloded from NCBI s Gene Expression Omnius (GEO). Degrdome reds were mpped to the extended pri-mirna hirpins. The positions of the degrdome reds represent unpped ends of in vivo RNA frgments. They were tllied on the sense strnd reltive to the hirpin nd plotted longside RNA seondry strutures nd the positions of mture mirna. Sequenes nd red numers for srnas in Aridopsis (68 dt sets) were downloded from GEO under the ession numers listed in Supplementry Tle 2 (ref. 4). Similrly, 45 srna deep-sequening dt sets in rie were retrieved from GEO nd re listed in Supplementry Tle 3. The srnas were mpped to mirna preursor sequenes, whih were downloded from mir dtse relese 6 using n in-house C++ progrm. We defined four riteri for potentil idiretionl proessing of pri-mirnas: (i) the srnas re proessed from 5 7 nt wy from rnhed or lrge terminl loops; (ii) there re no ler unstrutured regions t 5 7 nt wy from the srna remnnts in the opposite diretion (Supplementry Fig. 7); (iii) s mirna:mirna duplexes re not preisely proessed in mny ses nd hve some minor forms entered round the predominnt mirna:mirna duplexes, we defined the srnas tht re t lest 3 nt wy from n djent minor form of mirna:mirna s eing derived from proessing wy tht is different from produtive proessing (Supplementry Tles 2 nd 3); nd (iv) srnas re primrily 2 nt long nd re nd present in t lest three individul lirries. 44. Zhng, X., Grreton, V. & Chu, N.H. The AIP2 E3 ligse ts s novel negtive regultor of ABA signling y promoting ABI3 degrdtion. Genes Dev. 9, (05). nture struturl & moleulr iology doi:0.038/nsm.2646