AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins

ADWAITA PRASAD PARIDA, AMRAPALI SHARMA and ARUN KUMAR SHARMA

J. Biosci. 42(1), March 2017, © Indian Academy of Sciences

Supplementary material

Supplementary figure 1. Negative control for FRET analysis and FRET efficiency. (A) Subcellular localization of CFP-AtMBD6 along with YFP protein was performed in onion epidermal cells. YFP was present in both cytoplasm and nucleus. It did not show a pattern of localization in the nucleus, which was similar to that of the interacting partners. The energy transfer between CFP-AtMBD6 and YFP was calculated using acceptor photo-bleaching protocol. There is no significant decrease in pre-bleaching and post-bleaching intensity of YFP indicating that the energy transfer from CFP to YFP is negligible. (B) The FRET efficiency during interaction of CFP-AtMBD6 and other interacting proteins.

Supplementary figure 2. Sequence alignment of NTF2 and RRM domains containing proteins from Arabidopsis thaliana. Sequence alignment using protein sequences of genes containing NTF2 and RRM domains was done by Clustal-W method. All the eight proteins showed conserved amino acids sequence within the NTF2 and RRM domains. The NTF2 domain contains some conserved amino acids, e.g. F 85,377, 381, Y298, 290, 304, S 308, R 312, G 357,364,V 362, 392, Q 379,L 383, and D 395. The RNA Recognition Motif (RRM) contains conserved amino acids in L 1088, F 11013, 1104, 1128, 1131, G 1105, 111, 1200, I 1107 and A1143. The gene AT3G07250 is the largest protein that contains two NTF and four RRM domains.

Supplementary figure 3. Conservation of NTF2 and RRM domain containing protein sequences in flowering plants. Sequence alignment of proteins containing NTF2 and RRM domains from Arabidopsis thaliana (At; AED97406.1), Oryza sativa (Os; CAH66639.1), Vitis vinifera (Vv; XP_003634318.1), Hordeum vulgare (Hv;BAK04969.1), Populus trichocarpa (Pt; XP_002328636.1), Zea mays (Zm; DAA38277.1) and Triticum aestivum (Ta; AEP33645.1) was done by Clustal-W method. Sequence analysis suggests presence of similar proteins in flowering plants.

Supplementary table 1. List of NTF2 and RRM domain containing proteins in Arabidopsis

TAIR locus ID NCBI protein reference sequences

No. of amino acids

No. of EST

AT1G13730 NP_563932.1 428 25AT2G03640 NP_001189508.1 454 26AT3G25150 NP_001189969.1 587 120AT5G43960 NP_199209.1 450 59AT5G60980 NP_200906.2 460 40AT5G48650 NP_199676.2 458 14AT1G69250 NP_177085.1 427 71AT3G07250 NP_187381.2 1294 2

Supplementary table 2. List of NTF2 and RRM domain containing proteins in flowering plants

Species name Number of amino acids

Gene bank ID

Hordeumvulgare 543 BAK04969.1Zea mays 487 DAA38277.1Oryza sativa Indica Group 488 CAH66639.1Oryza sativa Japonica Group 486 NP_001052571.1Triticumaestivum 436 AEP33645.1Populustrichocarpa 393 XP_002328636.1Vitisvinifera 469 XP_003634318.1

Supplementary table 3. List of primers

Primers for cloningPrimer name SequencepSITE-MBD6 FCACCATGTCAGATTCTGTGGCCpSITE-MBD6 R AAAGCCGACACTTTACTAGGGTTTCpSITE-RPS2C F CACCATGGCCGAACGTGGTGGAG pSITE-RPS2C R AAAGGCTTGGTCTTCAACC pSITE-NTF2 F CACCATGGCACAGCAGGAAGCTAG pSITE-NTF2 R AAAAGATGAACCACCACCTCGAGpSITE-HDAC6 F CACCATGGAGGCAGACGAAAGCpSITE-HDAC6 R AAAAGACGATGGAGGATTCACGpGBK-MBD6 FCCCTGAATTCATGTCAGATTCTGTGGCCGGCGpGBK-MBD6 R GATGACGGATCCTCAAGCCGACACTTTACTAGGGpSITE-U2B F CACCATGTTAACGGCAGATATACCACpSITE-U2B R TCATTTCTTGGCGAAAGAGATGAC

Primers for methylation sensitive PCRsdc F CTCGCCTCCTCTCCGGACCAsdc R CGATGAGCCTGAGCAGCGAAmop9.5 F CCGGTTTTGCTGGTGTTCCGCmop9.5 R CTGGTCGAGCCGGTTGTCCA1G28002F TGGGAGATTCACAACCAAGGGGGA1G28002R AGCCTCTCTTCCACGGACATGCT2G13469 F ACCTCGCAGTTGGGGTCTAGCA2G13469 R TCAGGCGGTGGTTGGAGCCT18S rRNA F TGTGGCGGGGAATTGCCGTGA18S rRNA R GCGCGCCTGCTGCCTTCCTT180 bp repeats F ACCATCAAAGCCTTGAGAAGCA180 bp repeats R CCGTATGAGTCTTTGTCTTTGTATCTTCTACTIN 12 F ACGTCGGAGACGAGGCACAGTACTIN 12 R ACACCGGAAACGCTCTGCCCmiRNA164 F AATGGGTGAGTAACACTTGATGGmiRNA164 R AGGGGGAGAGACACGTGTTGmiRNA167 F CCAGTAGCAGTTAAGCTGCCAmiRNA167 R CATTAGCAGGTGAAACTACCAGCmiRNA319 F GCTTCCTTGAGTCCATTCACAGmiRNA319 R AGAGGGAGCTCCCTTCAGTCmiRNA842 F AAGCGGGATGATGAATCCGAmiRNA842 R AAGTGGGATGACGGATCTGACmiRNA159 F GAGCTCCTTAAAGTTCAAACATGmiRNA159 R TAGAGCTCCCTTCAATCCAAAGmiRNA828 F ACAAAGTCACAAAAGTGATAAACTCTCmiRNA828 R CTCATACATGATATTAAATAGTCCCAC