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
AT1G13730
AT2G03640
AT3G25150
AT5G43960
AT5G60980
AT5G48650
AT1G69250
AT3G07250
NCBI protein
reference sequences
NP_563932.1
NP_001189508.1
NP_001189969.1
NP_199209.1
NP_200906.2
NP_199676.2
NP_177085.1
NP_187381.2
No. of amino
acids
428
454
587
450
460
458
427
1294
No. of EST
25
26
120
59
40
14
71
2
Supplementary table 2. List of NTF2 and RRM domain containing proteins in flowering plants
Species name
Hordeumvulgare
Zea mays
Oryza sativa Indica Group
Oryza sativa Japonica Group
Triticumaestivum
Populustrichocarpa
Vitisvinifera
Number of
amino acids
543
487
488
486
436
393
469
Gene bank ID
BAK04969.1
DAA38277.1
CAH66639.1
NP_001052571.1
AEP33645.1
XP_002328636.1
XP_003634318.1
Supplementary table 3. List of primers
Primers for cloning
Primer name Sequence
pSITE-MBD6 FCACCATGTCAGATTCTGTGGCC
pSITE-MBD6 R
AAAGCCGACACTTTACTAGGGTTTC
pSITE-RPS2C F
CACCATGGCCGAACGTGGTGGAG
pSITE-RPS2C R
AAAGGCTTGGTCTTCAACC
pSITE-NTF2 F CACCATGGCACAGCAGGAAGCTAG
pSITE-NTF2 R AAAAGATGAACCACCACCTCGAG
pSITE-HDAC6 F
CACCATGGAGGCAGACGAAAGC
pSITE-HDAC6 R
AAAAGACGATGGAGGATTCACG
pGBK-MBD6 FCCCTGAATTCATGTCAGATTCTGTGGCCGGCG
pGBK-MBD6 R
GATGACGGATCCTCAAGCCGACACTTTACTAGGG
pSITE-U2B F CACCATGTTAACGGCAGATATACCAC
pSITE-U2B R TCATTTCTTGGCGAAAGAGATGAC
Primers for methylation sensitive PCR
sdc F CTCGCCTCCTCTCCGGACCA
sdc R CGATGAGCCTGAGCAGCGAA
mop9.5 F
CCGGTTTTGCTGGTGTTCCGC
mop9.5 R
CTGGTCGAGCCGGTTGTCCA
1G28002F
TGGGAGATTCACAACCAAGGGGGA
1G28002R
AGCCTCTCTTCCACGGACATGCT
2G13469 F
ACCTCGCAGTTGGGGTCTAGCA
2G13469 R
TCAGGCGGTGGTTGGAGCCT
18S rRNA F TGTGGCGGGGAATTGCCGTGA
18S rRNA R GCGCGCCTGCTGCCTTCCTT
180 bp repeats F
ACCATCAAAGCCTTGAGAAGCA
180 bp repeats R
CCGTATGAGTCTTTGTCTTTGTATCTTCT
ACTIN 12 F ACGTCGGAGACGAGGCACAGT
ACTIN 12 R ACACCGGAAACGCTCTGCCC
miRNA164 F AATGGGTGAGTAACACTTGATGG
miRNA164 R AGGGGGAGAGACACGTGTTG
miRNA167 F CCAGTAGCAGTTAAGCTGCCA
miRNA167 R CATTAGCAGGTGAAACTACCAGC
miRNA319 F GCTTCCTTGAGTCCATTCACAG
miRNA319 R AGAGGGAGCTCCCTTCAGTC
miRNA842 F AAGCGGGATGATGAATCCGA
miRNA842 R AAGTGGGATGACGGATCTGAC
miRNA159 F GAGCTCCTTAAAGTTCAAACATG
miRNA159 R TAGAGCTCCCTTCAATCCAAAG
miRNA828 F ACAAAGTCACAAAAGTGATAAACTCTC
miRNA828 R CTCATACATGATATTAAATAGTCCCAC