static-content.springer.com10.1007... · Web viewAtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins Adwaita
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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
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
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