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Structural basis for the modular recognition of single-stranded RNA by PPR proteins

LETTER

doi:10.1038/nature12651

Ping Yin 1,2*,Quanxiu Li 1,2*,Chuangye Yan 2,3,Ying Liu 1,2,Junjie Liu 2,3,Feng Yu 4,Zheng Wang 5,Jiafu Long 5,Jianhua He 4,Hong-Wei W ang 2,3,Jiawei W ang 1,2,Jian-Kang Zhu 6,7,Yigong Shi 2,3&Nieng Yan 1,2

Pentatricopeptide repeat (PPR)proteins represent a large family of sequence-specific RNA-binding proteins that are involved in mul-tiple aspects of RNA metabolism.PPR proteins,which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants 1–5,recognize single-stranded RNA (ssRNA)in a modular fashion 6–8.The maize chloroplast protein PPR10binds to two similar RNA sequences from the ATPI –ATPH and PSAJ –RPL33intergenic regions,referred to as ATPH and PSAJ ,respectively 9,10.By protecting the target RNA elements from 59or 39exonucleases,PPR10defines thecorresponding 59and 39messenger RNA termini 9–11.Despite rigorous functional characterizations,the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated.Here we report the crystal structures of PPR10in

RNA-free and RNA-bound states at resolutions of 2.85and 2.45A

?,respectively.In the absence of RNA binding,the nineteen repeats of PPR10are assembled into a right-handed superhelical spiral.PPR10forms an antiparallel,intertwined homodimer and exhibits conside-rable conformational changes upon binding to its target ssRNA,an 18-nucleotide PSAJ element.Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10repeats following the pre-dicted code.The molecular basis for the specific and modular recog-nition of RNA bases A,G and U is revealed.The structural elucidation of RNA recognition by PPR proteins provides an important frame-work for potential biotechnological applications of PPR proteins in RNA-related research areas.

PPR proteins function in multiple aspects of organelle RNA meta-bolism,such as RNA splicing,editing,degradation and translation 1–5.In plants,PPR mutants may cause embryonic lethality 12–14,and a number of PPR proteins act as restorers of fertility to overcome cytoplasmic male sterility 15–19.In humans,mutations in the mitochondrial PPR protein LRPPRC are associated with the French-Canadian-type Leigh syndrome characterized by the deficiency in Complex IV 20,21.

PPR proteins contain 2–30tandem repeats,each typically compris-ing 35amino acids that are organized into a hairpin of a -helices 1,6,22,23.PPRs are divided into two classes:the P-class,whose members only comprise the 35-amino-acid repeats;and the PLS-class,which has repeats of 31–36amino acids and extra domains at the carboxyl ter-minus 3,https://www.sodocs.net/doc/214749165.html,putational and biochemical analyses suggest that PPR proteins may recognize RNA in a modular fashion,but different from that of the RNA-binding PUF domain 6,24.The putative RNA recog-nition code by PPR proteins derived from bioinformatic and biochem-ical analyses awaits structural corroboration 2,6–8.

To elucidate the mechanism of specific RNA recognition by PPR proteins,we sought to determine the crystal structure of well-characterized PPR proteins in complex with their target RNAs.The recombinant protein of maize chloroplast PPR10,which belongs to the P-class,speci-fically binds to the 17-nucleotide (nt)(ATPH )and 18-nt (PSAJ )RNA

oligonucleotides (Extended Data Fig.1a)10.We launched a systematic effort to determine the structures of PPR10in both RNA-free and RNA-bound states.

The crystal structure of the RNA-free PPR10fragment (residues 61–786)containing quadruple Cys mutations (C256S/C279S/C430S/C449S)

was determined at 2.85A

?resolution.PPR10forms a right-handed two-turn superhelical assembly,with 19PPR motifs (residues107–771)capped by three short a -helices at the amino-terminal domain (NTD)and a single a -helix at the C terminus (Fig.1a).Capping motifs are known to contribute to ligand specificity for repeat proteins such as TPR (tetra-tricopeptide repeat)25and TALE (transcription activator-like effector)26,27.The function of the extra motifs in PPR10remains to be determined.The 35amino acids in each PPR motif form a hairpin of a -helices,each containing four helical turns,followed by a five-residue loop (Fig.1b).The two helices,designated helix a and helix b,are connected by a short turn of two amino acids.Helices a and b of each repeat constitute the inner and outer layers of the superhelical assembly,respectively (Fig.1a).In the crystals,there is one molecule of PPR10in each asymmetric unit,yet two symmetry-related molecules are intertwined in an antiparallel fashion.The N terminus of one molecule is in close contact with the C terminus of the other,yielding an overall appearance of an ellipsoid

with a polar axis of approximately 140A

?and an equatorial diameter of 70A

?(Fig.1c).On the basis of the PPR10structure,we defined the starting amino acid of helix a as the first residue in a PPR motif (Fig.1b and Extended Data Fig.1b).This definition results in a one-residue shift either forwards 6,12or backwards 7,28within each repeat compared to the previously described boundary of a PPR motif (Extended Data Fig.1c).With the new boun-dary assignment of a PPR motif,the residues that were predicted to determine RNA binding specificity are all included in one structurally intact motif.We hope that this structure-based demarcation of the PPR motif will simplify future descriptions of PPR proteins.

After numerous unsuccessful crystallization trials for PPR10–ATPH complexes,we finally determined the structure of PPR10(residues 69–786,C256S/C279S/C430S/C449S)in the presence of 18-nt PSAJ RNA

(59-GUAUUCUUUAAUUAUUUC-39)at 2.45A

?resolution (Extended Data Table1).In the crystals,there is one antiparallel PPR10dimer in each asymmetric unit.Analysis by sedimentation equilibrium analy-tical ultracentrifugation (SE-AUC)of PSAJ -bound PPR10(residues 37–786,C256S/C279S/C430S/C449S)supports its dimeric existence at micromolar concentration in solution (Extended Data Fig.2).The two PPR10protomers can be superimposed with a root-mean-squared

deviation of 1.3A

?over 629C a atoms (Extended Data Fig.3).The ove-rall appearance of the dimer has changed to a hollow cylindrical tube (Fig.2a),and the N-and C-terminal portions of the PPR10protomer

are compressed towards the centre,resulting in a reduction of 20A

?in axial length (Fig.2b).

*These authors contributed equally to this work.

State Key Laboratory of Bio-membrane and Membrane Biotechnology,Tsinghua University,Beijing 100084,China.2Center for Structural Biology,School of Life Sciences and School of Medicine,Tsinghua-Peking Center for Life Sciences,Tsinghua University,Beijing 100084,China.3Ministry of Education Key Laboratory of Protein Science,Tsinghua University,Beijing 100084,China.4Shanghai Institute of Applied Physics,Chinese Academy of Sciences,239Zhangheng Road,Shanghai 201204,China.5State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences,Nankai University,94Weijin Road,Tianjin 300071,China.6Shanghai Center for Plant Stress Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China.7

Department of Horticulture and Landscape Architecture,Purdue University,West Lafayette,Indiana 47907,USA.00M O N T H 2013|V O L 000|N A T U R E |1

Following assignment of most amino acids of PPR10into the elec-tron density map,strong electron densities indicative of RNA bases became clearly visible in the cavities on both ends of the cylindrical tube (Fig.2c).Assignment of 18and 14nucleotides of the two bound RNA elements was validated by the anomalous signals of bromine (Br),which were collected for crystals of PPR10bound to Br-labelled RNA oligonucleotides (Extended Data Fig.4and Extended Data Table 2).The 59and 39portions of the ssRNA are specifically recognized by the N-terminal repeats of one protomer and C-terminal repeats of the other.By contrast,the middle portion of the ssRNA,comprising nucle-otides U5to A10,remains largely uncoordinated by PPR10(Fig.2d and Extended Data Fig.5a,b).

PPR10has 19repeats and the bound PSAJ RNA contains 18nucleo-tides.Consistent with a bioinformatic prediction 6,specific recognition of the PSAJ RNA begins with repeat 3(Fig.3a).Each of the first four nucleotides on the 59end,59-GUAU-39,is recognized by one PPR10repeat.Such recognition exhibits a modular pattern involving residues that were predicted through biochemical and bioinformatic analyses 2,6,7.Each RNA base is surrounded by four residues,the 2nd residues from two adjacent repeats,and the 5th and 35th residues from a correspond-ing repeat.In addition to base recognition,the backbone phosphate or ribose groups of the bound PSAJ RNA are also coordinated by charged or polar amino acids from PPR10(Extended Data Fig.5c).

A polar amino acid located at the 5th position in each repeat appears to be the most important determinant for RNA base specificity.Thr 178,Asn 213,Ser 249and Asn 284in repeats 3–6recognize the bases G1,U2,A3and U4,respectively,through direct hydrogen bonds (Fig.3b).The importance of the 5th residue in RNA recognition is supported by mutational analysis.Mutating any of the 5th residues in repeats 4(N213A),5(S249L)or 6(N284A)resulted in complete abolishment of RNA binding.By contrast,substitution of the 5th residues of repeats 7,8,10,11or 13,which are not involved in RNA binding in the struc-ture,showed little or no effect on PSAJ binding (Extended Data Fig.6).Buttressing the hydrogen bonds,five residues at the 2nd position of PPR repeats 3–7sandwich the four bases mainly through van der Waals interactions (Fig.3a).For example,G1is surrounded by Arg 175/Val 210of repeats 3and 4.Similarly,U2,A3and U4are sandwiched by Val 210/Phe 246,Phe 246/Val 281and Val 281/Val 316,respectively (Fig.3).The 35th residue is located in the vicinity of the base.It is possible that water molecules,although invisible in the structure,may mediate hydrogen bonds between the polar residues and the bases.Importantly,Asp

244

140 ?

NTD

Helix a

Helix b

1a 7a

9a 10a

12a

14a

14b

13a

5a 16a 1

7a 18a

9a 1b

2b 3b

9b 10b 11b 13b 12b 17b 1

9b N

TD N

3a 4a

5a 6a 10a 12a 14a 14b 13a 15a 16a 17a 18a 19a 10b 11b 13b 12b 17b 19b NTD 1a 2a

1b C

9a 19b 19a

19b

Figure 1|Crystal structure of RNA-free PPR10.a ,Overall structure of

RNA-free PPR10.The fragment (residues 61–786,C256S/C279S/C430S/C449S)comprises 19repeats capped by a small NTD (light purple)and a C-terminal helix (yellow).The two helices within each repeats,designated helix a and helix b,are coloured green and blue,respectively.b ,Structural

superimposition of the 19repeats of PPR10.c ,Overall structure of the PPR10dimer.Two molecules from adjacent asymmetric units form an intertwined antiparallel dimer.All structure figures were prepared with PyMol 30.

a b

NTD

s s R

N A

s s R

N A NTD

120 ?

140 ?

Figure 2|Structure of PPR10bound to an 18-nt PSAJ RNA element.a ,The PPR10dimer (residues 69–786,C256S/C279S/C430S/C449S)forms a

cylindrical tube in the presence of PSAJ .The two protomers are coloured light purple and grey with their NTDs coloured blue and cyan.b ,The PPR10protomer undergoes pronounced conformational changes upon binding to PSAJ .The structure of RNA-free PPR10is coloured magenta with the NTD coloured lilac.c ,Electron densities found in the cavities on both ends of the PPR10dimer.The ‘omit’electron density,with a close-up view in the inset,is contoured at 3s .d ,Overall structure of the PPR10–PSAJ complex.The two ssRNA molecules are coloured yellow and orange.

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and Asp314,the35th residues in repeats4and6,are respectively hydrogen bonded to Asn213and Asn284,the5th residues in the corresponding repeats,and may help to stabilize their conformation for base recog-nition(Fig.3b and Extended Data Fig.6d).

Recognition of the39end of the PSAJ RNA by the C-terminal repeats in the other PPR10protomer appears to be less modular except for U15 and U16,which are coordinated by repeats16and17following the described recognition pattern for U2and U4(Fig.4a).The bases A11 and C18are coordinated in a non-modular fashion.The adenine base of A11donates a hydrogen bond to Asp630,the35th residue of repeat 15,whereas the cytosine base of C18makes a hydrogen bond to Ser714

on the last helical turn of helix18a(Fig.4b).A number of direct and water-mediated hydrogen bonds are found between the backbone phosphate/ribose groups of U15-U16-U17-C18and the polar residues on repeats15–19of PPR10(Fig.4c).

In the structure of PSAJ-bound PPR10,only six out of18nucleotides in the PSAJ RNA element strictly follow the modular pattern(Fig.4d). Two bases,A11and C18,are bound by PPR10in a non-modular fashion, and the other10bases are literally uncoordinated.Notably,only17 repeats in each PPR10protomer are available for the binding of18 nucleotides.It remains to be seen whether the17-nt ATPH binds to repeats3–19of PPR10in a completely modular fashion.Binding of ATPH leads to dissociation of the PPR10dimer(Extended Data Fig.2)6,10. Interestingly,analysis by SE-AUC suggests that,although RNA-free PPR10forms a stable dimer,PSAJ binding weakens the PPR10dimer formation(Extended Data Fig.2).It remains to be investigated whether PSAJ-bound PPR10is a dimer under physiological conditions,in which the protein concentration can be very low.Nevertheless,the crystal structure of PPR10bound to the18-nt PSAJ RNA element reveals the molecular basis for specific recognition between a PPR protein and its target RNA sequence.

Recognition of the six RNA nucleotides59-G1-U2-A3-U4-39and 59-U15-U16-39by repeats3–6and repeats16and17,respectively,largely supports the predicted code for base discrimination in ssRNA,where the bases G,U and A are specifically recognized by the5th residues of a PPR motif:Thr,Asn and Ser,respectively6,7.The2nd and35th residues, sitting in the vicinity of the bases,contribute to RNA binding(Fig.4d and Extended Data Fig.6c,d).The prediction that base C is recognized by an Asn at the5th position can also be conveniently rationalized based on our crystal structure(Extended Data Fig.7).One unexpected feature is that the2nd residues from two consecutive repeats sandwich one base;therefore the identity of the2nd residue on the next repeat must be considered for RNA binding.Base sandwiching by hydro-phobic residues or Arg is also observed in the recognition of ssRNA by PUF proteins24,29,although PPR and PUF proteins exhibit distinct RNA binding modes(Extended Data Fig.8).

Further biochemical,computational,structural and in vivo charac-terizations are required to completely rationalize the codes for specific RNA recognition by PPRs and to engineer PPR proteins for targeted RNA manipulations.The structures reported here provide unprece-dented insights into the recognition mechanism of RNA elements by PPR proteins and serve as an important foundation for understanding the function and mechanism of numerous PPR proteins in RNA meta-bolism,and for the potentially customized design of specific-RNA-binding PPR proteins.

METHODS SUMMARY

Thecodon-optimized complementaryDNAoffull-length PPR10(GeneID:100302579) from Zea mays was subcloned into pET15b vector(Novagen).Overexpression of PPR10protein was induced in Escherichia coli BL21(DE3).To crystallize PPR10, we mounted a systematic protein engineering effort including a series of protein truncations and mutations of Cys residues.There are18Cys residues within the repeat region.We generated18mutants,each consisting of a single Cys to Ser muta-tion and tested their binding with the17-nt ATPH element.For those that com-pletely retained binding affinity,we further grouped them to double,triple and quadruple mutations.Finally,the PPR10mutant containing C256S/C279S/C430S/ C449S showed the same binding affinity as wild type and exhibited excellent protein behaviour.All the PPR10proteins used in the manuscript contain the quadruple Cys mutations.The RNA-free PPR10fragment(residues61–786,C256S/C279S/ C430S/C449S)was eventually crystallized in the space group P21212.The structure

R175

V210

F246V281 T178

N213

S249

V210

F246

D244

F246

V281

S279 V210

T208

b NTD

3a4a5a

5′

3′

R175

V210

F246V281

V316 T178

N213

S249

V210

F246

D244

F246

V281

S279 V210

T208

5′

U2A3

Figure3|Base-specific recognition of ssRNA by PPR10repeats.a,The four nucleotides at the59end of the PSAJ RNA segment are specifically recognized in a modular fashion.Inset:each of the four RNA bases at the59end is sandwiched by two residues at the2nd positions of adjacent repeats.b,Specific recognition of the bases G,U and A by PPR10repeats.The side chain of the5th residue in each repeat,which makes a direct hydrogen bond to the base,is highlighted in cyan.The hydrogen bonds are represented by red dotted lines.

Residue no.

within repeat

U15

A11

C18

U16

I632

D666

C701

V668

V703

19a

18a

17a

16a

15a

16a

18a

15a

17a

18a

C18

U17

U16

U15

N635

N671

S714

D630

S604

S636T672N675

R742

U15

A11

C18

U16

I632

D666

C701

V668

V703

19a

18a

17a

16a

15a

16a

18a

15a

17a

18a

C18

U17

U16

U15

3′

N635

N671

S714

D630

S604

S636T672N675

R742 b c

′

PSAJ

ATPH

′

PPR10 repeat no. 3 5 7 9 11 13 15 17 19

U12

A10U13

A14

U15

U16

19a

18a

17a

16a

15a

14a U17

C18

A11

U12

A10

U13

A14

U15

U16

19a

18a

17a

16a

15a

14a U17

C18

Figure4|Coordination of the39-end segment of the PSAJ RNA by PPR10. a,Recognition of the bases U15and U16by PPR10follows the code discussed in Fig.3.b,The bases A11and C18are hydrogen bonded to polar residues on PPR10in a non-modular fashion.c,The backbone of the39-end segment of PSAJ is coordinated through direct or water-mediated hydrogen bonds.

d,Summary of specific recognition of PSAJ RNA by PPR10.Left,the residues at the2nd,5th and35th positions of PPR10motifs and the corresponding sequences of the target RNA elements.The structurally corroborated recognition codes are shaded yellow.The difference in RNA sequences of PSAJ and ATPH is shaded grey.Right,the structure of repeats3–19of PPR10with their2nd(yellow),5th(cyan)and35th(wheat)residues shown in spheres,and the six recognized RNA bases shown in orange.

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was determined by selenium-based single-wavelength anomalous diffraction and refined to2.85A?resolution(Extended Data Table1).In the effort to crystallize PPR10in complex with its target RNA,despite numerous trials,most PPR10–ATPH complexes defied crystallization;for those that crystallized,X-ray diffrac-tion was consistently poor.We applied the same strategy to complexes between PPR10and PSAJ.After screening more than100,000conditions,we were able to crystallize the complex between PPR10(residues69–786,C256S/C279S/C430S/ C449S)and the18-nt PSAJ RNA(59-GUAUUCUUUAAUUAUUUC-39)in the space group P43.These crystals diffract X-rays beyond2.5A?.The structure was determined by molecular replacement using successive segments of the RNA-free PPR10structure,but not the entire molecule.We were able to assign all18nucleo-tides of one bound PSAJ RNA element,but only14of the other.For details of elec-trophoretic mobility shift assay and SE-AUC experiments,please refer to Methods. Online Content Any additional Methods,Extended Data display items and Source Data are available in the online version of the paper;references unique to these sections appear only in the online paper.

Received28May;accepted12September2013.

Published online27October2013.

1.Small,I.D.&Peeters,N.The PPR motif—a TPR-related motif prevalent in plant

organellar proteins.Trends Biochem.Sci.25,45–47(2000).

2.Nakamura,T.,Yagi,Y.&Kobayashi,K.Mechanistic insight into pentatricopeptide

repeat proteins as sequence-specific RNA-binding proteins for organellar RNAs in plants.Plant Cell Physiol.53,1171–1179(2012).

3.Schmitz-Linneweber,C.&Small,I.Pentatricopeptide repeat proteins:a socket set

for organelle gene expression.Trends Plant Sci.13,663–670(2008).

4.Fujii,S.&Small,I.The evolution of RNA editing and pentatricopeptide repeat

genes.New Phytol.191,37–47(2011).

5.Kotera,E.,Tasaka,M.&Shikanai,T.A pentatricopeptide repeat protein is essential

for RNA editing in chloroplasts.Nature433,326–330(2005).

6.Barkan,A.et al.A combinatorial amino acid code for RNA recognition by

pentatricopeptide repeat proteins.PLoS Genet.8,e1002910(2012).

7.Yagi,Y.,Hayashi,S.,Kobayashi,K.,Hirayama,T.&Nakamura,T.Elucidation of the

RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants.PLoS ONE8,e57286(2013).

8.Yagi,Y.et al.Pentatricopeptide repeat proteins involved in plant organellar RNA

editing.RNA Biol.10,1236–1242(2013).

9.Pfalz,J.,Bayraktar,O.A.,Prikryl,J.&Barkan,A.Site-specific binding of a PPR

protein defines and stabilizes59and39mRNA termini in chloroplasts.EMBO J.28, 2042–2052(2009).

10.Prikryl,J.,Rojas,M.,Schuster,G.&Barkan,A.Mechanism of RNA stabilization and

translational activation by a pentatricopeptide repeat protein.Proc.Natl Acad.Sci.

USA108,415–420(2011).

11.Zhelyazkova,P.et al.Protein-mediated protection as the predominant mechanism

for defining processed mRNA termini in land plant chloroplasts.Nucleic Acids Res.

40,3092–3105(2012).

12.Lurin,C.et al.Genome-wide analysis of Arabidopsis pentatricopeptide repeat

proteins reveals their essential role in organelle biogenesis.Plant Cell16,

2089–2103(2004).

13.Cushing,D.A.,Forsthoefel,N.R.,Gestaut,D.R.&Vernon,D.M.Arabidopsis emb175

and other ppr knockout mutants reveal essential roles for pentatricopeptide repeat (PPR)proteins in plant embryogenesis.Planta221,424–436(2005).

14.Khrouchtchova,A.,Monde,R.A.&Barkan,A.A short PPR protein required for the

splicing of specific group II introns in angiosperm chloroplasts.RNA18,

1197–1209(2012).15.Bentolila,S.,Alfonso,A.A.&Hanson,M.R.A pentatricopeptide repeat-containing

gene restores fertility to cytoplasmic male-sterile plants.Proc.Natl https://www.sodocs.net/doc/214749165.html,A 99,10887–10892(2002).

16.Desloire,S.et al.Identification of the fertility restoration locus,Rfo,in radish,as a

member of the pentatricopeptide-repeat protein family.EMBO Rep.4,588–594 (2003).

17.Wang,Z.et al.Cytoplasmic male sterility of rice with boro II cytoplasm is caused by

a cytotoxic peptide and is restored by two related PPR motif genes via distinct

modes of mRNA silencing.Plant Cell18,676–687(2006).

18.Chase,C.D.Cytoplasmic male sterility:a window to the world of plant

mitochondrial–nuclear interactions.Trends Genet.23,81–90(2007).

19.Hu,J.et al.The rice pentatricopeptide repeat protein RF5restores fertility in Hong-

Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162.Plant Cell24,109–122(2012).

20.Mootha,V.K.et al.Identification of a gene causing human cytochrome c oxidase

deficiency by integrative genomics.Proc.Natl https://www.sodocs.net/doc/214749165.html,A100,605–610

(2003).

21.Ruzzenente,B.et al.LRPPRC is necessary for polyadenylation and coordination of

translation of mitochondrial mRNAs.EMBO J.31,443–456(2012).

22.Howard,M.J.,Lim,W.H.,Fierke,C.A.&Koutmos,M.Mitochondrial ribonuclease

P structure provides insight into the evolution of catalytic strategies for

precursor-tRNA59processing.Proc.Natl https://www.sodocs.net/doc/214749165.html,A109,16149–16154

(2012).

23.Ringel,R.et al.Structure of human mitochondrial RNA polymerase.Nature478,

269–273(2011).

24.Wang,X.,McLachlan,J.,Zamore,P.D.&Hall,T.M.Modular recognition of RNA by a

human pumilio-homology domain.Cell110,501–512(2002).

25.Grove,T.Z.,Cortajarena,A.L.&Regan,L.Ligand binding by repeat proteins:

natural and designed.Curr.Opin.Struct.Biol.18,507–515(2008).

26.Deng,D.et al.Structural basis for sequence-specific recognition of DNA by TAL

effectors.Science335,720–723(2012).

27.Gao,H.,Wu,X.,Chai,J.&Han,Z.Crystal structure of a TALE protein reveals an

extended N-terminal DNA binding region.Cell Res.22,1716–1720(2012). 28.Kobayashi,K.et al.Identification and characterization of the RNA binding surface

of the pentatricopeptide repeat protein.Nucleic Acids Res.40,2712–2723(2012).

29.Filipovska,A.&Rackham,O.Modular recognition of nucleic acids by PUF,TALE

and PPR proteins.Mol.Biosyst.8,699–708(2012).

30.DeLano,W.L.The PyMOL Molecular Graphics System.https://www.sodocs.net/doc/214749165.html,

(2002).

Acknowledgements We thank X.Yu and Y.Chen at the Institute of Biophysics,Chinese Academy of Sciences,for technical support.We thank K.Hasegawa and T.Kumasaka at the SPring-8beamline BL41XU for on-site assistance.This work was supported by funds from the Ministry of Science and Technology(grant number2011CB910501for N.Y.),and Projects91017011(N.Y.),31070644(N.Y.),31021002(Y.S.,N.Y.,J.W.)and 31200567(P.Y.)of the National Natural Science Foundation of China.The research of N.Y.was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.

Author Contributions P.Y.,Q.L.,J.-K.Z.,Y.S.and N.Y.designed all experiments.P.Y.,Q.L., C.Y.,Y.L.,J.L.,F.Y.,Z.W.,J.L.,J.H.,H.-W.W.,J.W.and N.Y.performed the experiments.All authors analysed the data and contributed to manuscript preparation.N.Y.wrote the manuscript.

Author Information The atomic coordinates and structure factors of RNA-free and RNA-bound PPR10have been deposited in the Protein Data Bank(PDB)with the accession codes4M57and4M59,respectively.Reprints and permissions information is available at https://www.sodocs.net/doc/214749165.html,/reprints.The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to N.Y.

(nyan@https://www.sodocs.net/doc/214749165.html,).

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METHODS

Protein preparation.The codon-optimized complementary DNA of full-length PPR10(Gene ID:100302579)from Zea mays was subcloned into pET15b vector (Novagen).Overexpression of PPR10protein was induced in E.coli BL21(DE3) with0.2mM isopropyl-b-D-thiogalactoside at an OD600nm of1.2.After growing for16h at16u C,the cells were collected,homogenized in a buffer containing25 mM Tris-HCl,pH8.0,and150mM NaCl.After sonication and centrifugation,the supernatant was applied to Ni21affinity resin(Ni-NTA,Qiagen)and further frac-tionated by ion-exchange chromatography(Source15Q,GE Healthcare).The PPR10mutants were generated using two-step PCR and subcloned,overexpressed and purified in the same way as the wild-type protein.

A systematic protein engineering effort was mounted for crystallization of RNA-free and-bound PPR10.A series of protein truncations were tested without giving rise to crystals.There are18Cys residues within the repeat region.It is well known that the presence of surface Cys residues,which are subject to oxidation,may lead to protein heterogeneity and impede crystallization.We therefore generated18 mutants,each consisting of a single Cys to Ser mutation and tested their binding with the17-nt ATPH element.For those that completely retained binding affinity, we further grouped them to double,triple and quadruple mutations.Finally,the PPR10mutant containing C256S/C279S/C430S/C449S showed the same binding affinity as wild type and exhibited excellent protein behaviour.For consistency,all the PPR10proteins used in the manuscript contain the quadruple Cys mutations. For the crystallization trials of RNA-free PPR10(residues61–786,C256S/C279S/ C430S/C449S),the protein was concentrated and applied to gel filtration chromato-graphy(Superdex-20010/30,GE Healthcare)in the buffer containing25mM Tris-HCl,pH8.0,150mM NaCl and10mM dithiothreitol(DTT).Selenomethionine (Se-Met)-derived protein was purified similarly.

To obtain the crystals of protein–RNA complex,PPR10(residues69–786,C256S/ C279S/C430S/C449S)was purified through Ni21affinity resin(Ni-NTA,Qiagen), followed by heparin affinity column(HiPrep Heparin FF16/10,GE Healthcare). The protein was then applied to gel filtration chromatography(Superdex-20010/30, GE Healthcare).The buffer for gel filtration contained25mM Tris-HCl,pH8.0, 50mM NaCl,5mM MgCl2and10mM DTT.The peak fractions were incubated with target RNA oligonucleotides with a molar ratio of approximately1:1.5at4u C for about40min before crystallization trials.

Crystallization.Both RNA-free and RNA-bound PPR10proteins were crystal-lized by hanging-drop vapour-diffusion method at18u C.PPR10(residues61–786, C256S/C279S/C430S/C449S),at a concentration of approximately6.0mg ml21, was mixed with an equal volume of reservoir solution containing1.8–2.1M sodium formate,and0.1M Bis-Tris propane,pH6.5.Plate-shaped crystals appeared over-night and grew to full size within1–2weeks.Se-Met-labelled protein was crystal-lized similarly.

To obtain crystals of protein–RNA complex,various combinations of protein boundaries and RNA oligonucleotides(Takara)were examined.Because the first visible residue in the structure of RNA-free PPR10starts at position69,we invested more effort into this construct.Finally,the protein(residues69–786,C256S/C279S/ C430S/C449S)and18-nt RNA from the PSAJ–RPL33intergenic region with the sequence59-GUAUUCUUUAAUUAUUUC-39(designated PSAJ RNA)gave rise to crystals in the reservoir solution containing8–10%(w/v)polyethylene glycol 3350,8%Tacsimate,pH6.0(Hampton Research),and0.1M MES,pH5.5. Data collection and structural determination.All data sets were collected at SSRF beamline BL17U or SPring-8beamline BL41XU and processed with the HKL2000 packages31.Further processing was carried out with programs from the CCP4suite32. Data collection and structure refinement statistics are summarized in Extended Data Tables1and2.

The RNA-free PPR10structure was solved by single anomalous diffraction (SAD)of Se-Met using the program ShelxC/D/E33.Then a crude helical model was manually built in the program https://www.sodocs.net/doc/214749165.html,ing this partial model as input,the identified Se atom positions were refined and phases were recalculated using the SAD experimental phasing module of the program Phaser35.With the improved map,the molecular boundary was unambiguously defined and one molecule was found inan asymmetry unit.Thecrudemodel wasfurtherrebuiltwithCoot and refined with Phenix36.The sequence docking was aided by anomalous map of selenium. Data sets collected from five crystals of the PPR10–RNA complex were merged for complete and better data.The structure of the PPR10–RNA complex was solved by molecular replacement with the newly solved RNA-free structure as the search model using the program Phaser35.To find the right solution,the structure of the RNA-free PPR10protomer was divided into three consecutive segments.The assign-ment of RNA sequence was aided by the anomalous signal of bromine obtained for crystals of PPR10in complex with Br-labelled RNA oligonucleotides,where U4/ U7/U15,U5/U7/U15or U12were substituted by5-bromouracil(Extended Data Table2).The structure was manually refined with Coot and Phenix iteratively (Extended Data Table1).

Electrophoretic mobility shift assay(EMSA).The ssRNA oligonucleotides were radiolabelled at the59end with[c-32P]ATP(PerkinElmer)catalysed by T4poly-nucleotide kinase(Takara).The sequences of ssRNA oligonucleotides used in EMSA are:PSAJ,59-GUAUUCUUUAAUUAUUUC-39;and ATPH,59-GUAUCCUUA ACCAUUUC-39.

For EMSA,PPR10(residues37–786,C256S/C279S/C430S/C449S)and the other variants consisting of the indicated point mutations were incubated with approximately40pM32P-labelled probe in the final binding reactions containing 40mM Tris-HCl,pH7.5,100mM NaCl,4mM DTT,0.1mg ml21BSA,5m g ml21 heparin and10%glycerol at room temperature(22u C)for20min.Reactions were then resolved on6%native acrylamide gels(37.5:1for acrylamide:bisacrylamide) in0.53Tris-glycine buffer under an electric field of15V cm21for40min.Vacuum-dried gels were visualized on a phosphor screen(Amersham Biosciences)with a Typhoon Trio Imager(Amersham Biosciences).

SE-AUC.The oligomeric states of PPR10(residues37–786,C256S/C279S/C430S/ C449S)with or without target RNA oligonucleotides in solution were investigated by AUC experiments.SE-AUC experiments were performed in a Beckman Coulter XL-I analytical ultracentrifuge using six-channel centrepieces.RNA-free PPR10,PSAJ-bound PPR10and ATPH-bound PPR10were in solutions contain-ing25mM Tris-HCl,pH8.0,150mM NaCl and2mM DTT.The sequences of RNA oligonucleotides were identical to those used in EMSA.Data were collected by interference detection at4u C for all three protein concentrations(4m M,6m M and8m M)at different rotor speeds(6,000,8,500and12,000r.p.m.).The buffer composition(density and viscosity)and protein partial specific volume(V-bar) were obtained using the SEDNTERP program(available through the Boston Biomedical Research Institute).The SE-AUC data were globally analysed using the Sedfit and Sedphat programs37and were fitted to a monomer–dimer equilib-rium model to determine the dissociation constants(K d)for the homodimers.

31.Otwinowski,Z.&Minor,W.Processing of X-ray diffraction data collected in

oscillation mode.Methods Enzymol.276,307–326(1997).

32.Collaborative Computational Project,Number4.The CCP4suite:programs for

protein crystallography.Acta Crystallogr.D50,760–763(1994).

33.Schneider,T.R.&Sheldrick,G.M.Substructure solution with SHELXD.Acta

Crystallogr.D58,1772–1779(2002).

34.Emsley,P.&Cowtan,K.Coot:model-building tools for molecular graphics.Acta

Crystallogr.D60,2126–2132(2004).

35.McCoy,A.J.et al.Phaser crystallographic software.J.Appl.Crystallogr.40,

658–674(2007).

36.Adams,P.D.et al.PHENIX:building new software for automated crystallographic

structure determination.Acta Crystallogr.D58,1948–1954(2002).

37.Schuck,P.On the analysis of protein self-association by sedimentation velocity

analytical ultracentrifugation.Anal.Biochem.320,104–124(2003).

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Extended Data Figure1|Sequence alignment of the19repeats of PPR10. a,PPR10from maize specifically recognizes two RNA elements.The cartoon above illustrates the predicted domain organization of PPR10.1and19refer to the repeat numbers.CTP,chloroplast transit peptide.The blue brick with‘?’represents a fragment of approximately30amino acids whose function remains to be characterized.The minimal RNA elements of PSAJ and ATPH that are targeted by PPR10are shown below the cartoon.b,Sequence alignment of19 repeats in PPR10.The secondary structural elements of a typical PPR motif are shown above.The residues at the2nd,5th and35th positions which were predicted to be the molecular determinants for RNA-binding specificity are highlighted in magenta.The RNA sequences that can be recognized by PPR10 are listed on the right,59to39from top to bottom.The nucleotides which are recognized by PPR10in a modular fashion in the PSAJ–PPR10structure are shaded grey.c,The three numbering systems for a PPR motif.1is being used by Lurin et al.12,Barkan et al.6and others;2is adopted by the Pfam database and being used by Kobayashi et al.28,Yagi et al.7and others;and3is our proposed, structure-based numbering system.The residues that are predicted to specifically recognize RNA are coloured magenta.

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Extended Data Figure2|AUC-SE of PPR10(residues37–786,C256S/

C279S/C430S/C449S)in the absence or presence of the target RNA elements.The molar concentrations of PPR10are indicated above each panel.PPR10and the RNA oligonucleotides were mixed at a stoichiometric ratio of approximately1:1.5.Details of the experiments are described in Methods.

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Extended Data Figure3|The two protomers of the RNA-bound PPR10 dimer exhibit similar conformations.a,The two protomers can be superimposed with a root-mean-squared deviation of1.31A?over629C a atoms.b,c,The two ssRNA segments are coordinated by the PPR10dimer similarly.The59and39segments of the bound PSAJ RNA are separately coordinated by the N-terminal repeats of one protomer(b),and the C-terminal repeats of the other protomer(c).Stereo-views are shown for all panels.

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Extended Data Figure4|Electron density maps for a bound ssRNA segment.a,The2F o–F c electron density for one segment of the bound PSAJ RNA.The electron density,contoured at1s and coloured blue,is displayed in stereo.b,c,The anomalous signals for bromine in the structures where the highlighted nucleotides were substituted with5-bromouracil(5-BrU).The anomalous signals,shown in magenta mesh,are contoured at5s.

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Extended Data Figure5|Coordination of the bound ssRNA by PPR10. a,The59and39portions of the PSAJ RNA element are separately bound by the N-terminal and C-terminal repeats of the two PPR10protomers.Shown here is a close-up view of the binding of PSAJ by one end of the PPR10dimer.

b,The nucleotides U5–A10,which form a U-turn in the ssRNA,are uncoordinated in the cavity of the PPR10dimer.The two protomers of PPR10 are shown in semi-transparent surface contour.c,The RNA backbone is coordinated by polar or charged residues through hydrogen bonds.The hydrogen bonds are represented by red dotted lines.The two protomers of PPR10are coloured light purple and grey.

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Extended Data Figure6|Mutational analysis of PPR10residues that may be important for PSAJ.a,EMSA analysis of the interaction between

PPR10(residues37–786,C256S/C279S/C430S/C449S)and PSAJ

(59-GUAUUCUUUAAUUAUUUC-39).PPR10was added with increasing concentrations of0,2,4,8,16,31,63,125,250,500,1,000nM in lanes1–11with approximately40pM32P-labelled PSAJ in each lane.b,Mutational analysis of the5th residues of the indicated PPR motifs.The indicated point mutations were introduced to PPR10(residues37–786,C256S/C279S/C430S/C449S). c,Examination of the2nd residues in repeats3and5.d,Examination of the 35th residue of repeat6.Note that the side group of Asp314is hydrogen bonded to the side chain of Asn284,the5th residue of repeat6.The same structural feature is also seen in repeat4(Fig.3b).

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Extended Data Figure 7|The predicted coordination of base C by an Asn at the 5th position of a PPR motif.Left,the coordination of base U by Asn observed in the structure.Right,the coordination of base C by Asn at the 5th position of a PPR motif modelled on the basis of the structure shown on the left.

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Extended Data Figure8|Comparison of ssRNA coordination by PUF and PPR proteins.a,The structure of the human PUF protein PUM1(also known as HSPUM)bound to the RNA element NRE1-19(PDB accession code,

1M8W)24.The PUF repeats constitute an arc with8-nt ssRNA bound to the concave side.Notably,the orientations of the bound RNA and the protein are antiparallel,namely the59end is close to the C terminus of PUF.b,The structure of a PUF repeat.One canonical PUF repeat contains three helices, of which a short helix precedes a helical hairpin.c,Representative recognition of the RNA bases G,U,A by PUF repeats as seen in the structure of PUM1 bound to NRE1-19.The amino acids are labelled by the repeat number

(r5,r6,r7,r8)followed by its one-letter code and position on the2nd helix within a PUF repeat(S3,N4,E8,and so on).The same scheme applies

to d.d,The coordination of RNA bases G,U,A by PPR10.It is noteworthy that PUF and PPR proteins share several common features for RNA binding:

(1)the ssRNA elements are coordinated by the helices on the inner layer;and

(2)the base is sandwiched mostly by hydrophobic residues or Arg.Yet the differences are evident between the two families of repeat proteins.As seen in c,the RNA base is usually coordinated by two residues that are located at the4th and the7th positions on helix2within a PUF repeat.By contrast,the base

is mainly coordinated by the5th residue of a PPR motif.The35th residue,the last residue of a PPR motif that is located at a loop region preceding the next PPR motif,also contributes to base recognition.

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Extended Data Table 1|Statistics of data collection and refinement.Values in parentheses are for the highest resolution

shell

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Extended Data Table 2|Statistics of data

collection

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Structural basis for the modular recognition of single-stranded RNA by PPR proteins

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