MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing

Transcription

1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing Junjie Yan 1, Qunxia Zhang 1, Zeyuan Guan 1, Qiang Wang 1, Li Li 1, Fengying Ruan 1, Rongcheng Lin 2, Tingting Zou 1 and Ping Yin 1 * 1 National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan , China. 2 Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing , China. These authors contributed equally to this work. * yinping@mail.hzau.edu.cn NATURE PLANTS DOI: /nplants Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 Supplementary Figure 1. Consensus sequence design for (PLS) 3 PPR. a, Conservation analysis of the P-, L-, and S-type motifs. Sequence logos were generated using repeats derived from all Arabidopsis thaliana PLS-type PPR proteins in WebLogo 3 ( The amino acids are colored based on the physiochemical properties of their side chains: small (G and A) in black, nucleophilic (S, T, and C) in blue, hydrophobic (V, L, I, M, and P) in green, aromatic (F, Y, and W) in red, acidic (D and E) in pink, amide (N and Q) in magenta, and basic (H, K and R) in orange. b, The complete sequence of the designer (PLS) 3 PPR. The nine tandem repeats containing 3 PLS triplets are shaded cyan. The N- and C-termini are capped. c, Purification of designer (PLS) 3 PPR. Upper panel: Gel-filtration chromatography. Lower panel: the purified protein examined by SDS-PAGE. 1

3 Supplementary Figure 2. Stereo overview of (PLS) 3 PPR. Symmetry mates of the PLS triplets from neighboring unit cells are shown. The full 9-repeat (PLS) 3 PPR protein containing 3 PLS triplets is highlighted in blue and gold. 2

4 Supplementary Figure 3. Structural alignment of the designer PLS-type (PLS) 3 PPR with the designer P-type PPR (PDB: 4WSL). (PLS) 3 PPR and P-type PPR are colored cyan and gray, respectively. 3

5 Supplementary Figure 4. Structural superimposition of P-, L- and S-type motifs of (PLS) 3 PPR. a, Alignment of the three P-type motifs. b, Alignment of the three L-type motifs. c, Alignment of the three S-type motifs. 4

6 Supplementary Figure 5. Multiple sequence alignment of the central MORF domains in Arabidopsis MORF family members. Secondary structural elements are indicated above the sequences. Residues involved in hydrophobic and hydrogen bond interactions are highlighted with blue and red balls, respectively. Residue numbers are indicated to the left of each sequence. Sequence alignment was carried out using MultiAlign. 5

7 Supplementary Figure 6. RNA-binding ability of MORF9, (PLS) 3 PPR, and (PLS) 3 PPR-MORF9 complex against by EMSA. The final protein concentrations in lanes 1-5 (MORF9), 6-10 ((PLS) 3 PPR), and ((PLS) 3 PPR-MORF9 complex) are 0, 4.5, 9, 18, and 36 M, respectively. B, bound; U, unbound. 6

8 Supplementary Figure 7. ITC profiles of RNA-binding ability of MORF9, (PLS) 3 PPR, and (PLS) 3 PPR-MORF9 complex. The RNA probe used is 5 -UAUUAUUAU-3. 7

9 Supplementary Figure 8. Interactions between MORF9 and the PLS triplets. a, Size-exclusion chromatography analysis of MORF9, (PLS) 1 PPR, (PLS) 2 PPR, (PLS) 1 PPR-MORF9, and (PLS) 2 PPR-MORF9. Fractions with the same elution volume from each injection were subjected to SDS-PAGE. b, The molar ratio of the complex was measured by analytical ultracentrifugation. Upper panel: c(s) distributions from the SV runs for (PLS) 1 PPR-MORF9, (PLS) 2 PPR-MORF9, and (PLS) 3 PPR-MORF9. Lower panel: the theoretical and experimental molecular weights of each complex. c, ITC profiles of the titration of (PLS) 1 PPR with MORF9. The single mutation D164K in MORF9 or K29D (L-type motif) in (PLS) 1 PPR disrupts the interactions. 8

10 Supplementary Figure 9. Structural alignment of free MORF9 with (PLS) 3 PPR-bound MORF9. a, Alignment of free MORF9 with the N-terminal (PLS) 3 PPR-bound MORF9. b, Alignment of free MORF9 with the middle (PLS) 3 PPR-bound MORF9. c, Alignment of free MORF9 with the C-terminal (PLS) 3 PPR-bound MORF9. MORF9 and (PLS) 3 PPR-bound MORF9 are colored cyan and gray, respectively. 9

11 Supplementary Figure 10. MORF9 interacts with the PLS-type PPR, LPA66, and increases its RNA-binding ability. a, Co-expression analysis of LPA66 and MORF9. Motif organization of LPA66 is depicted as rectangle box. The designations of the P, L, S, E, and DYW domain correspond to those proposed by Lurin et al. (2004). The labeled number from 51 to 510 excludes the signal peptide sequence and the DYW domain. The MBP-tagged PLS repeats and E domain of LPA66 (Residues ) was co-expressed with untagged MORF9 (Residues ), and purified to homogeneity. b, Proteins used for the following EMSA assay. Asterisk indicates a C-terminal 8 His-tagged MORF9 (Residues ), the molecular weight of which is slightly higher than the untagged MORF9. c-e, RNA-binding ability was examined for MORF9 (c), LPA66 (d), and LPA66-MORF9 complex (e). The final protein concentrations of MORF9, LPA66 and LPA66-MORF9 complex in lanes 1-10 are 0, 0.016, , , 0.125, 0.25, 0.5, 1, 2, and 4 M, respectively. f, Titration experiment 10

12 for LPA66 with the increased amounts of MORF9. The final protein concentration of LPA66 in each lane is 2 M. The final protein concentrations of MORF9 in lane 1-5 are 0, 2, 4, 8, and 16 M, respectively. The psbf RNA probe is 5 -UAGCUGUACCUACCGUUUCUUUU-3. B, bound; U, unbound. 11

13 Supplementary Table 1. Data collection, phasing and refinement statistics (PLS) 3 PPR SeMet (PLS) 3 PPR (PDB: 5IZW) MORF9 SeMet MORF9 (PDB: 5GI0) (PLS) 3 PPR-MORF9 (PDB: 5IWW) Data collection Space group P4 3 P4 3 C121 C121 P6 2 Cell dimensions a, b, c (Å) 67.37, 67.37, , 67.41, , 50.69, , 50.69, , , ( o ) 90, 90, 90 90, 90, 90 90, , 90 90, , 90 90, 90, 120 Resolution (Å) ( ) ( ) ( ) ( ) ( ) Rmerge(%) 10.3 (17.1) 6.1 (107.0) 11.9 (29.8) 11.9 (29.8) 7.1 (57.7) I/ (I) 39.1 (20.1) 24.1 (2.1) 32.6 (10.5) 32.6 (10.5) 15.1 (3.1) Completeness (%) 99.9 (99.8) 99.9 (97.6) 99.5 (99.4) 99.5 (99.4) 98.5 (99.9) Redundancy 17.4 (17.7) 13.4 (10.5) 8.9 (8.8) 8.9 (8.8) 5.6 (5.7) Refinement Resolution (Å) No. reflections 5,532 26,305 9,121 9,121 25,740 Rwork/Rfree (%) 17.85/ / /22.08 No. atoms Protein 1, ,931 Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles ( o ) Values in parentheses are for the highest resolution shell. R merge =Σ h Σ i I h,i -I h /Σ h Σ i I h,i, where I h is the mean intensity of the i observations of symmetry related reflections of h. R=Σ F obs -F calc /ΣF obs, where F calc is the calculated protein structure factor from the atomic model (R free was calculated with 5% of the reflections selected). 12