Supplementary Figure 1 - Nature

3.0

1.0 0

24

48 72 Time (hr)

Vector

MajSAT

-tubulin -tubulin

Merge

3.0 1.0

MajSAT 0

96

Dapi

Vector

24

48 72 Time (hr)

96

f Micronuclei

8

0

5.0

e

*

(%) 16

c

7.0

Anaphase bridge

(%) 10

*

5

0

Vector MajSAT

Percentage of Anaphase bridge

b

5.0

Percentage of micronuclei

d

Percentage of mitotic anomaries

Cell proliferation rate (fold)

a

Cell proliferation rate (fold)

Supplementary Figure 1

(%) 4

* 2

0

Vector

MajSAT

Supplementary Figure 1 | MajSAT expression causes chromosomal instability. a, b, The cell growth curve did not significantly change with MajSAT RNA expression. a, Blue: K512-vector; red: K512-EF1α-MajSAT. b, K512-TREtight-MajSAT cells. Blue: mock red: doxycycline treatment. Data represent the mean ± s.e. of four independent experiments. c, Representative images of multi-polar mitosis in K512-EF1α-MajSAT (MajSAT) cells. α-tubulin indicates microtubule. γ-tubulin indicates centrosome. d, Percentage of multi-polar mitotic cells in all mitotic cells in K512-EF1α-MajSAT (MajSAT) cells. Mitotic cells were counted, and the ratio of abnormal mitosis was determined out of 200 mitotic cells counted. Data represent the mean ± s.e. of four independent experiments. *: p < 0.05. e, Representative images of micronuclei (left) and anaphase bridge (right) in K512-EF1α-majSAT (MajSAT) cells. f, Percentage of interphase cells with micronuclei and anaphase bridge was calculated out of the 500 cells counted. Data represent the mean ± s.e. of four independent experiments. *: p < 0.05.

Supplementary Figure 2 Dloop-amplified seq 512L-dloop-c48-T7 512L-dloop-c49-T7 512L-dloop-c50-T7 512L-dloop-c51-T7 512L-dloop-c52-T7 512L-dloop-c53-T7 512L-dloop-c54-T7 512L-dloop-c55-T7 512L-dloop-c56-T7 512L-dloop-c57-T7 512L-dloop-c58-T7 512L-dloop-c59-T7 512L-dloop-c60-T7 512L-dloop-c61-T7 512L-dloop-c62-T7 512L-dloop-c64-T7 512L-dloop-c65-T7 512L-dloop-c66-T7 512L-dloop-c67-T7 512L-dloop-c68-T7 512L-dloop-c69-T7 512L-dloop-c70-T7 512L-dloop-c71-T7 512L-dloop-c72-T7 512L-dloop-c73-T7 512L-dloop-c74-T7 512L-dloop-c75-T7 512L-dloop-c76-T7 512L-dloop-c77-T7 512L-dloop-c78-T7 512L-dloop-c79-T7 512L-dloop-c80-T7 512L-dloop-c81-T7 512L-dloop-c82-T7 512L-dloop-c83-T7 512L-dloop-c84-T7 512L-dloop-c85-T7 512L-dloop-c86-T7 512L-dloop-c87-T7 512L-dloop-c88-T7 blank 512L-dloop-c1-T7 512L-dloop-c2-T7 512L-dloop-c3-T7 512L-dloop-c4-T7 512L-dloop-c5-T7 512L-dloop-c6-T7 512L-dloop-c8-T7 512L-dloop-c9-T7 512L-dloop-c10-T7 512L-dloop-c11-T7 512L-dloop-c13-T7 512L-dloop-c14-T7 512L-dloop-c15-T7 512L-dloop-c16-T7 512L-dloop-c17-T7 512L-dloop-c18-T7 512L-dloop-c20-T7 512L-dloop-c21-T7 512L-dloop-c22-T7 512L-dloop-c23-T7 512L-dloop-c24-T7

1 84 86 85 86 86 84 85 85 84 84 85 85 85 85 85 84 85 84 84 85 85 85 87 86 84 85 84 84 86 86 84 85 84 84 84 84 85 85 87 85 19 89 86 84 84 83 90 90 84 85 85 86 89 86 84 85 84 88 85 87 89 89

-------TCTTTTTATTTTGGCCTACTTTCATCAACATAGCCGTCAAGGCATGAAAGGACAGCACACAGTCTAGACGCACCTACGGTGAA TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC.................................................................................G. TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... -----------------------------------------------------------------------------------------TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC................................................................................... TGGATCC...................................................................................

83 173 175 174 175 175 173 174 174 173 173 174 174 174 174 174 173 174 173 173 174 174 174 176 175 173 174 173 173 175 175 173 174 173 173 173 173 174 174 176 174 19 178 175 173 173 172 179 179 173 174 174 175 178 175 173 174 173 177 174 176 178 178

Dloop-amplified seq 512L-dloop-c48-T7 512L-dloop-c49-T7 512L-dloop-c50-T7 512L-dloop-c51-T7 512L-dloop-c52-T7 512L-dloop-c53-T7 512L-dloop-c54-T7 512L-dloop-c55-T7 512L-dloop-c56-T7 512L-dloop-c57-T7 512L-dloop-c58-T7 512L-dloop-c59-T7 512L-dloop-c60-T7 512L-dloop-c61-T7 512L-dloop-c62-T7 512L-dloop-c64-T7 512L-dloop-c65-T7 512L-dloop-c66-T7 512L-dloop-c67-T7 512L-dloop-c68-T7 512L-dloop-c69-T7 512L-dloop-c70-T7 512L-dloop-c71-T7 512L-dloop-c72-T7 512L-dloop-c73-T7 512L-dloop-c74-T7 512L-dloop-c75-T7 512L-dloop-c76-T7 512L-dloop-c77-T7 512L-dloop-c78-T7 512L-dloop-c79-T7 512L-dloop-c80-T7 512L-dloop-c81-T7 512L-dloop-c82-T7 512L-dloop-c83-T7 512L-dloop-c84-T7 512L-dloop-c85-T7 512L-dloop-c86-T7 512L-dloop-c87-T7 512L-dloop-c88-T7 blank 512L-dloop-c1-T7 512L-dloop-c2-T7 512L-dloop-c3-T7 512L-dloop-c4-T7 512L-dloop-c5-T7 512L-dloop-c6-T7 512L-dloop-c8-T7 512L-dloop-c9-T7 512L-dloop-c10-T7 512L-dloop-c11-T7 512L-dloop-c13-T7 512L-dloop-c14-T7 512L-dloop-c15-T7 512L-dloop-c16-T7 512L-dloop-c17-T7 512L-dloop-c18-T7 512L-dloop-c20-T7 512L-dloop-c21-T7 512L-dloop-c22-T7 512L-dloop-c23-T7 512L-dloop-c24-T7

84 174 176 175 176 176 174 175 175 174 174 175 175 175 175 175 174 175 174 174 175 175 175 177 176 174 175 174 174 176 176 174 175 174 174 174 174 175 175 177 175 19 179 176 174 174 173 180 180 174 175 175 176 179 176 174 175 174 178 175 177 179 179

GAATCATTAGTCCGCAAAACCCAATCACCTAAGGCTAATTATTCATGCTTGTTAGACATAAATGCTACTCAATACCAAATTTTAACTCTC .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... -----------------------------------------------------------------------------------------.......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... ...................................................................................G...... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... ..........................................................................................

173 263 265 264 265 265 263 264 264 263 263 264 264 264 264 264 263 264 263 263 264 264 264 266 265 263 264 263 263 265 265 263 264 263 263 263 263 264 264 266 264 19 268 265 263 263 262 269 269 263 264 264 265 268 265 263 264 263 267 264 266 268 268


174 264 266 265 266 266 264 265 265 264 264 265 265 265 265 265 264 265 264 264 265 265 265 267 266 264 265 264 264 266 266 264 265 264 264 264 264 265 265 267 265 19 269 266 264 264 263 270 270 264 265 265 266 269 266 264 265 264 268 265 267 269 269

CAAACCCCCCACCCCCTCCTCTTAATGCCAAACCCCAAAAACACTAAGAACTTGAAAGACATATAATATTAACTATCAAACCCTATGTCC .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... -----------------------------------------------------------------------------------------.......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .........................C................................................................ .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... ..........................................................................................

263 353 355 354 355 355 353 354 354 353 353 354 354 354 354 354 353 354 353 353 354 354 354 356 355 353 354 353 353 355 355 353 354 353 353 353 353 354 354 356 354 19 358 355 353 353 352 359 359 353 354 354 355 358 355 353 354 353 357 354 356 358 358


264 354 356 355 356 356 354 355 355 354 354 355 355 355 355 355 354 355 354 354 355 355 355 357 356 354 355 354 354 356 356 354 355 354 354 354 354 355 355 357 355 19 359 356 354 354 353 360 360 354 355 355 356 359 356 354 355 354 358 355 357 359 359

TGATCAATTCTAGTAGTTCCCAAAATATGACTTATATTTTAGTACTTGTAAAAATTTTACAAAATCATGTTCCGTGAACCAAAACTCTAA .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... ..............................................C........................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... -----------------------------------------------------------------------------------------.......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... .......................................................................................... ..........................................C...............................................

353 443 445 444 445 445 443 444 444 443 443 444 444 444 444 444 443 444 443 443 444 444 444 446 445 443 444 443 443 445 445 443 444 443 443 443 443 444 444 446 444 19 448 445 443 443 442 449 449 443 444 444 445 448 445 443 444 443 447 444 446 448 448


354 444 446 445 446 446 444 445 445 444 444 445 445 445 445 445 444 445 444 444 445 445 445 447 446 444 445 444 444 446 446 444 445 444 444 444 444 445 445 447 445 19 449 446 444 444 443 450 450 444 445 445 446 449 446 444 445 444 448 445 447 449 449

TCATACTCTATTACGCAATAAACATTAACAAGTTAATGTAGCTTAATAACAAAGCAAAGCACTGAAAATGCTTAGATG-----------..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC -----------------------------------------------------------------------------------------..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC ..............................................................................GGATCCGAGCTC

Supplementary Figure 2 | Multiple alignment of the sequences in the D-loop region of mitochondrial DNA in K512-vector cells. Sequences in the D-loop region from control cells (K512-vector) are shown. The top line is the reference sequences: mouse consensus mitochondrial DNA (GenBank NC_005089.1, bases 15916-16299; 1-48). Mutated bases are indicated with frames.

431 533 535 534 535 535 533 534 534 533 533 534 534 534 534 534 533 534 533 533 534 534 534 536 535 533 534 533 533 535 535 533 534 533 533 533 533 534 534 536 534 19 538 535 533 533 532 539 539 533 534 534 535 538 535 533 534 533 537 534 536 538 538

Supplementary Figure 3 Dloop-amplified 512LM-dloop-c1-T7 512LM-dloop-c2-T7 512LM-dloop-c3-T7 512LM-dloop-c5-T7 512LM-dloop-c6-T7 512LM-dloop-c8-T7 512LM-dloop-c11-T7 512LM-dloop-c12-T7 512LM-dloop-c13-T7 512LM-dloop-c14-T7 512LM-dloop-c15-T7 512LM-dloop-c16-T7 512LM-dloop-c17-T7 blank 512LM-dloop-c54-T7 512LM-dloop-c55-T7 512LM-dloop-c56-T7 512LM-dloop-c57-T7 512LM-dloop-c58-T7 512LM-dloop-c59-T7 512LM-dloop-c60-T7 512LM-dloop-c61-T7 512LM-dloop-c62-T7 512LM-dloop-c63-T7 512LM-dloop-c64-T7 512LM-dloop-c65-T7 512LM-dloop-c66-T7 512LM-dloop-c67-T7 512LM-dloop-c68-T7 512LM-dloop-c69-T7 512LM-dloop-c70-T7 512LM-dloop-c71-T7 512LM-dloop-c72-T7 512LM-dloop-c73-T7 512LM-dloop-c74-T7 512LM-dloop-c75-T7 512LM-dloop-c76-T7 512LM-dloop-c77-T7 512LM-dloop-c78-T7 512LM-dloop-c79-T7 512LM-dloop-c80-T7 512LM-dloop-c81-T7 512LM-dloop-c82-T7 512LM-dloop-c83-T7 512LM-dloop-c84-T7 512LM-dloop-c85-T7 512LM-dloop-c86-T7 512LM-dloop-c87-T7 512LM-dloop-c88-T7 512LM-dloop-c89-T7 512LM-dloop-c90-T7 512LM-dloop-c91-T7 512LM-dloop-c92-T7 512LM-dloop-c93-T7 512LM-dloop-c94-T7

1 77 78 80 78 79 77 78 77 76 75 75 76 76 5 75 76 75 75 77 77 75 77 76 76 75 78 78 76 75 77 76 76 81 75 75 77 78 77 77 77 81 76 75 77 77 77 77 75 76 79 77 79 78 75 78

----------------TCTTTTTATTTTGGCCTACTTTCATCAACATAGCCGTCAAGGCATGAAAGGACAGCACACAGTC CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ -------------------------------------------------------------------------------CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC...........A.................................................... CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC............A................................................... CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................ CTGGACTAGTGGATCC................................................................

64 156 157 159 157 158 156 157 156 155 154 154 155 155 5 154 155 154 154 156 156 154 156 155 155 154 157 157 155 154 156 155 155 160 154 154 156 157 156 156 156 160 155 154 156 156 156 156 154 155 158 156 158 157 154 157

Dloop-amplified 512LM-dloop-c1-T7 512LM-dloop-c2-T7 512LM-dloop-c3-T7 512LM-dloop-c5-T7 512LM-dloop-c6-T7 512LM-dloop-c8-T7 512LM-dloop-c11-T7 512LM-dloop-c12-T7 512LM-dloop-c13-T7 512LM-dloop-c14-T7 512LM-dloop-c15-T7 512LM-dloop-c16-T7 512LM-dloop-c17-T7 blank 512LM-dloop-c54-T7 512LM-dloop-c55-T7 512LM-dloop-c56-T7 512LM-dloop-c57-T7 512LM-dloop-c58-T7 512LM-dloop-c59-T7 512LM-dloop-c60-T7 512LM-dloop-c61-T7 512LM-dloop-c62-T7 512LM-dloop-c63-T7 512LM-dloop-c64-T7 512LM-dloop-c65-T7 512LM-dloop-c66-T7 512LM-dloop-c67-T7 512LM-dloop-c68-T7 512LM-dloop-c69-T7 512LM-dloop-c70-T7 512LM-dloop-c71-T7 512LM-dloop-c72-T7 512LM-dloop-c73-T7 512LM-dloop-c74-T7 512LM-dloop-c75-T7 512LM-dloop-c76-T7 512LM-dloop-c77-T7 512LM-dloop-c78-T7 512LM-dloop-c79-T7 512LM-dloop-c80-T7 512LM-dloop-c81-T7 512LM-dloop-c82-T7 512LM-dloop-c83-T7 512LM-dloop-c84-T7 512LM-dloop-c85-T7 512LM-dloop-c86-T7 512LM-dloop-c87-T7 512LM-dloop-c88-T7 512LM-dloop-c89-T7 512LM-dloop-c90-T7 512LM-dloop-c91-T7 512LM-dloop-c92-T7 512LM-dloop-c93-T7 512LM-dloop-c94-T7

65 157 158 160 158 159 157 158 157 156 155 155 156 156 5 155 156 155 155 157 157 155 157 156 156 155 158 158 156 155 157 156 156 161 155 155 157 158 157 157 157 161 156 155 157 157 157 157 155 156 159 157 159 158 155 158

TAGACGCACCTACGGTGAAGAATCATTAGTCCGCAAAACCCAATCACCTAAGGCTAATTATTCATGCTTGTTAGACATAA ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ .............................C.................................................. ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ .....................................G.......................................... -------------------------------------------------------------------------------................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ............................................................................G... ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................

144 236 237 239 237 238 236 237 236 235 234 234 235 235 5 234 235 234 234 236 236 234 236 235 235 234 237 237 235 234 236 235 235 240 234 234 236 237 236 236 236 240 235 234 236 236 236 236 234 235 238 236 238 237 234 237


145 237 238 240 238 239 237 238 237 236 235 235 236 236 5 235 236 235 235 237 237 235 237 236 236 235 238 238 236 235 237 236 236 241 235 235 237 238 237 237 237 241 236 235 237 237 237 237 235 236 239 237 239 238 235 238

ATGCTACTCAATACCAAATTTTAACTCTCCAAA-CCCCCCACCCCCTCCTCTTAATGCCAAACCCCAAAAACACTAAGAA .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. -------------------------------------------------------------------------------.................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................C.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-.............................................. .................................-..............................................

223 315 316 318 316 317 315 316 315 314 313 313 314 314 5 313 314 313 313 315 315 313 315 314 314 313 316 316 314 313 315 314 314 319 313 313 315 316 315 315 316 319 314 313 315 315 315 315 313 314 317 315 317 316 313 316


224 316 317 319 317 318 316 317 316 315 314 314 315 315 5 314 315 314 314 316 316 314 316 315 315 314 317 317 315 314 316 315 315 320 314 314 316 317 316 316 317 320 315 314 316 316 316 316 314 315 318 316 318 317 314 317

CTTGAAAGACATATAATATTAACTATCAAACCCTATGTCCTGATCAATTCTAGTAGTTCCCAAAATATGACTTATATTTT ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ -------------------------------------------------------------------------------................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................

303 395 396 398 396 397 395 396 395 394 393 393 394 394 5 393 394 393 393 395 395 393 395 394 394 393 396 396 394 393 395 394 394 399 393 393 395 396 395 395 396 399 394 393 395 395 395 395 393 394 397 395 397 396 393 396


304 396 397 399 397 398 396 397 396 395 394 394 395 395 5 394 395 394 394 396 396 394 396 395 395 394 397 397 395 394 396 395 395 400 394 394 396 397 396 396 397 400 395 394 396 396 396 396 394 395 398 396 398 397 394 397

AGTACTTGTAAAAATTTTACAAAATCATGTTCCGTGAACCAAAACTCTAATCATACTCTATTACGCAATAAACATTAACA ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ -------------------------------------------------------------------------------................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ...G............................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................ ................................................................................

383 475 476 478 476 477 475 476 475 474 473 473 474 474 5 473 474 473 473 475 475 473 475 474 474 473 476 476 474 473 475 474 474 479 473 473 475 476 475 475 476 479 474 473 475 475 475 475 473 474 477 475 477 476 473 476


384 476 477 479 477 478 476 477 476 475 474 474 475 475 5 474 475 474 474 476 476 474 476 475 475 474 477 477 475 474 476 475 475 480 474 474 476 477 476 476 477 480 475 474 476 476 476 476 474 475 478 476 478 477 474 477

AGTTAATGTAGCTTAATAAC-AAAGCAAAGCACTG-AAAATGCTTAGATG-----------------------------....................-..............-...........-..GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T -------------------------------------------------------------------------------....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................A..............A..............GGATCCGAGCCTCGGTACCAAGCTTAAGTT ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGNACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T ....................-..............-..............GGATCCGAG-CTCGGTACCAAGCTTAAG-T

431 550 552 554 552 553 551 552 551 550 549 549 550 550 5 549 550 549 549 551 551 549 551 550 550 549 552 552 550 549 551 550 550 559 549 549 551 552 551 551 552 555 550 549 551 551 551 551 549 550 553 551 553 552 549 552

Supplementary Figure 3 | Multiple alignment of the sequences in the D-loop region of mitochondrial DNA in K512-EF1αMajSAT cells. Sequences in the D-loop region from K512-EF1α-MajSAT cells are shown. The top line is the reference sequences. Mutated bases are surrounded with frames. Deleted bases are indicated by “-”.

Supplementary Figure 4 Dapi

RNase

MALAT1

Merge

Dapi

MALAT1

Merge

Dapi

PPIB

Merge

Dapi

PPIB

Merge

Dapi

MajSAT

Merge

Dapi

MajSAT

Merge

-

+

Supplementary Figure 4 | MajSAT RNA is distributed primarily in the cytoplasm. K512 cells were transiently transfected with pLVSIN-EF1α-MajSAT plasmid. MajSAT RNA was detected by in situ hybridization. MALAT1 RNA, a control for nuclear localization, and PPIB RNA, a control for cytoplasmic localization, were also evaluated. RNase-treated cells served as a negative control. Representative images from three independent experiments are shown. Bar: 10 μm.

Supplementary Figure 5 a

Dapi

YBX1

Merge

Vector

MajSAT

UV irradiation (40J/m2)

b

Dapi

MajSAT

YBX1

Merge

UV

Supplementary Figure 5 | Nuclear translocation of YBX1 is impaired in MajSAT RNA-expressing cells after UV irradiation. a, YBX1 translocated into the nucleus after 40 J/m2 UV irradiation in K512 cells with control vector, whereas in stably MajSAT RNA-expressing cells, YBX1 remained in the cytoplasm. Representative cell images of three independent experiments are shown. Bar: 10 μm. b, MajSAT RNA co-localized with YBX1 and inhibited nuclear transportation in UV irradiated cells. K512 cells transiently transfected with pLVSIN-EF1 α-MajSAT plasmid were irradiated at 40 J/m2 UV. After 6 h, MajSAT RNA and YBX1 were double stained by in situ hybridization and immunofluorescence staining. A MajSAT RNA-expressing cell and untransfected cells are intentionally shown in the same field of view for comparison. Representative cell images from three independent experiments are shown. Bar: 10 μm.

Supplementary Figure 6 a Vector

MajSAT Fw Rv

MajSAT antisense probe MajSAT sense probe

b (nt) 1,000

Dapi

MajSAT Rv

Merge

1,000

Actb

1,800

c Dapi

YBX1

Merge

Untreat

H2O2

Supplementary Figure 6 | Cytoplasmic retention of YBX1 does not occur after expression of antisense MajSAT RNA. a, Antisense MajSAT RNA expression. K512 cells transiently transfected with sense MajSAT RNA (MajSAT Fw) or antisense MajSAT RNA (MajSAT Rv) expression constructs were used for northern blotting. The membrane was probed with sense and antisense MajSAT RNA probes and then re-probed with β-actin (Actb). nt, nucleotides. Representative results from three independent experiments are shown. b, Antisense MajSAT RNA was expressed primarily in the nucleus. K512 cells were transiently transfected with pLVSIN-EF1 α-MajSAT-Rv plasmid. Antisense MajSAT RNA was detected by in situ hybridization. Representative images from five independent experiments are shown. Bar: 10 μm. c, YBX1 was translocated into the nucleus in antisense MajSAT RNA-expressing cells following H2O2 stimulation. K512 cells stably expressing antisense MajSAT RNA were treated with or without 300 μM H2O2 for 6 h, and YBX1 immunostaining was performed. Representative images from five independent experiments are shown. Bar: 10 μm.


Dapi

RBBP6

Merge

Dapi

RBBP6

Merge

Vector

MajSAT H2O2

b

0 Dapi

CTCF

6 Merge

Dapi

CTCF

(hr) Merge

Vector

MajSAT H2O2

c

0 Dapi

ANKRD2

6 Merge

Dapi

ANKRD2

(hr) Merge

Vector

MajSAT H2O2

d

0 Dapi

p53

6 Merge

Dapi

p53

(hr) Merge

Vector

MajSAT H2O2

0

6

(hr)

Supplementary Figure 7 | Intracellular localization of YBX1-interacting proteins in MajSAT RNA-expressing cells. K512 control (vector) and MajSAT RNA stably expressing K512 cells (MajSAT) were treated with or without 300 μM H2O2 for 6 h. YBX1-interacting proteins, RBBP6 (a), CTCF1 (b), ANKRD2 (c), and p53 (d), were subjected to immunostaining to visualize their intracellular localization. Translocation of RBBP6 into the nucleus following H2O2 treatment was partially impaired in MajSAT RNA-expressing cells. Representative images from three independent experiments are shown. Bar: 10 μm.


YBX1 Vector GFP

b Dapi

(kDa)

GFP

YBX1

75 YBX1

YBX1-GFP in K512

50

50

ACTB

c

Vector

mtDNA-encoded gene (%)

MajSAT MajSAT+YBX1 200

*

*

*

*

100

0

mt-Co1

mt-Cytb

Supplementary Figure 8 | Forced YBX1 expression rescues MajSAT RNA-induced impaired function of YBX1. a, Western blotting of K512 cells stably overexpressing YBX1-GFP chimeric protein. The upper band (approximately 78 kDa) represents ectopic YBX1-GFP and the lower bands (approximately 48 kDa) represent endogenous YBX1 protein. Representative results of two independent experiments are shown. b, Immunofluorescent images of K512 stably expressing YBX1-GFP cells. YBX1 antibody reacts with both endogenous YBX1 and ectopic YBX-GFP. Representative cell images are shown. Bar: 10 μm. c, Mitochondria encoded genes (mt-Co1 and mt-Cytb) in K512 cells were quantified by quantitative PCR. The levels reflect mitochondrial DNA damages. Values from control cells were set as 100, and the values from the cells with MajSAT RNA expression with and without forced YBX1 expression were determined. Data represent the mean ± s.e. of four independent experiments. *: p < 0.05.


b Vector

MajSAT -double

MajSAT

(nt)

2.0

MajSAT

8-OHdG level (ng/ml)

2,000 1,000 500 Actb

Vector MajSAT MajSAT-double * *

1.0

1,800 0 H2O2

c

0

2

24

(hr)

d

16

* *

* 8

0 MNU

*

*

Mock

Vector MajSAT MajSAT-double

2 week

mtDNA-encoded gene (%)

6-TG resistant colonies

Vector MajSAT MajSAT-double

150

* *

* *

100

50

0

mt-Co1

mt-Cytb

Supplementary Figure 9 | MajSAT RNA containing six basic units exhibits similar effects as those of MajSAT RNA containing three basic units. a, MajSAT RNA expression in K512 cells stably transduced with constitutively expressing constructs. MajSAT contains three basic units, while MajSAT-double contains longer sequences of six basic units. The same membrane was re-probed with β-actin (Actb). Nt: nucleotides. b, Recovery from oxidative DNA damage was delayed in a similar manner in MajSAT RNA-expressing cells (MajSAT) and MajSAT-double RNA-expressing cells (MajSAT-double). Levels of 8-OHdG were determined by competitive ELISA following H2O2 treatment for the indicated periods. Data represent the mean ± s.e. of three independent experiments. *, p < 0.05. c, Spontaneous missense mutations in the HPRT gene were increased in MajSAT-double cells similarly as MajSAT cells. Cells were treated with 2.0 μM MNU for 2 weeks to promote the tendency for mutation and subsequently cultured in 6-TG-containing media. The number of surviving colonies, which presumably developed mutations in the HPRT gene, was counted. Data represent the mean ± s.e. of three independent experiments. *, p < 0.05. d, Mitochondrial damage was similarly induced in MajSAT and MajSAT-double cells. Mitochondrially encoded genes (mt-Co1 and mt-Cytb) in K512 cells were quantified by quantitative PCR. The levels reflect mitochondrial DNA damage. Values from the control cells (vector) were set a 100%. Data represent the mean ± s.e. of four independent experiments. *, p < 0.05.


b YBX1

1

48 A/P

124

323 a.a.

217

CSD

c (kDa)

CTD

A: Flag

-

d

IP:

e

A

B C

D

MajSAT

Anti-flag

siRNA

Endo YBX1

FmYBX1

- A B C D D IP: Anti-flag IgG

0.2 0 H2O2

+

MajSAT MajSAT+FmYBX1+siYBX1 6-TG resistant colonies

*

24

-

g

*

2

RT(-) MajSAT

0.4

*

0

lnput (5%) IP: Anti-flag IgG

*

MajSAT MajSAT+FmYBX1+siYBX1 1.6

- NC #1 #2

IgG

- A BC D - ABC D D

MajSAT MajSAT+FmYBX1+siYBX1 * 0.6

-

+ + + +

(nt) 200

(hr)

*

32

16

0 MNU

*

Mock

2 week

h

MajSAT MajSAT+FmYBX1+siYBX1 mtDNA-encoded gene (%)

 Absorbance of BER activity

Dapi

+ + + + +

FmYBX1 -

D

MajSAT

8-OHdG level (ng/ml)

Endo YBX1

20

D: Flag

0 H2O2

37 25

C: Flag

0.8

FmYBX1

ACTB

B: Flag

f

50

500

* *

400 300 200 100 0

mt-Co1

mt-Cytb

Supplementary Figure 10 | YBX1 that no longer interacts with MajSAT RNA is functional in MajSAT RNA-expressing cells. a, Deletion constructs of the YBX1 protein. Plasmids expressing C terminal domain-deleted and flag-tagged YBX1 were constructed as indicated (constructs A, B, C, and D). A/P: alanine and proline domain, CSD: cold shock domain, CTD: C-terminal domain, aa: amino acids. b, (Upper panel) 293TN cells were transiently transfected with flag-tagged YBX1-expressing plasmids as indicated, and cell lysates containing synthesized MajSAT RNA were subjected to immunoprecipitation (IP) with anti-flag antibodies. IP using isotype IgG with construct D was performed as a negative control. (Lower panels) The binding of MajSAT RNA to flag-tagged YBX1 proteins was determined by RT-PCR. Semi-quantitative RT-PCR images of MajSAT RNA are shown. Five percent of the cell lysate was used as a control (input). As a negative control, samples that were not subjected to reverse transcription were used and presented in the lower panel (RT(-)). Representative results from two independent experiments are shown. c, Replacement of endogenous YBX1 with the flag-tagged C-terminally deleted YBX1 (FmYBX1) that no longer interacts with MajSAT RNA. K512 cells stably expressing MajSAT RNA and FmYBX1 protein were transfected with siRNAs targeting the 5’ untranslated region (#1) and exon 7 of YBX1 (deleted in construct C) (#2) to knock down endogenous YBX1 protein expression. NC: negative control siRNA. Protein levels of endogenous and mutant YBX1 were determined by western blotting using anti-C terminal YBX1 (recognizing only endogenous YBX1) and anti-flag antibodies (recognizing only mutant YBX1). The membrane was also probed with β-actin (ACTB) to confirm approximately equal protein loading. Representative results from two independent experiments are shown. d, FmYBX1 protein can translocate to the nucleus following H2O2 treatment. K512 cells stably expressing MajSAT RNA and FmYBX1 protein were treated with 300 μM H2O2 for 6 h. YBX1 was immunostained with the anti-flag antibody (green), which recognizes FmYBX1. Representative images from three independent experiments are shown. Bar: 10 μm. e, BER activity was recovered by replacing wild-type YBX1 with FmYBX1 in MajSAT RNA-expressing and endogenous YBX1 knock down cells (MajSAT + FmYBX1 + siYBX1). 8-OHdG excision activity was determined using a colorimetric DNAzyme assay. To determine the response to oxidative stress, cells were incubated in 400 μM H2O2 for 4 h. siRNA #1 in (c) was used for siYBX1. Data represent the mean ± s.e. of two independent experiments. *, p < 0.05. f, The recovery of oxidative DNA damage was restored by replacing wild-type YBX1 with FmYBX1 in MajSAT RNA-expressing and endogenous YBX1 knock down cells (MajSAT + FmYBX1 + siYBX1). Levels of 8-OHdG following H2O2 treatment for the indicated periods were determined by competitive ELISA. Data represent the mean ± s.e. of three independent experiments. *, p < 0.05. g, Spontaneous missense mutations in the HPRT gene were decreased in MajSAT + FmYBX1 + siYBX1 cells. Cells were treated with 2.0 μM MNU for 2 weeks to promote the tendency for mutation and subsequently cultured in 6-TG-containing media. The number of surviving colonies, which presumably developed mutations in the HPRT gene, was counted. Data represent the mean ± s.e. of three independent experiments. *, p < 0.05. h, Mitochondrial damage was recovered in MajSAT + FmYBX1 + siYBX1 cells. The expression of mitochondrially encoded genes (mt-Co1 and mt-Cytb), whose levels reflect mitochondrial damage, was quantified in K512 cells by quantitative PCR. Values from cells expressing MajSAT RNA only (MajSAT) were set at 100%. Data represent the mean ± s.e. of four independent experiments. *, p < 0.05.

Supplementary Figure 11 Figure 2b

Figure 3b HNRNP-U

NCL

SYNCRIP

IGF2BP1

YBX1

HNRNP-B1/A2

majSAT

Actb

HNRNP-A1

Figure 3c HA

RT-PCR

Supplementary Figure 11 | Full-length blot images for the main figures. Full-length northern blotting, western blotting, and gel images for Figure 2b, Figure 3b, and Figure 3c.

Band

Protein name

C1 (115kDa)

Heterogenous nuclear ribonucleoprotein U (Hnrnp U) Protein bicaudal C homolog 1 Bicaudal C homolog 1 (Drosophila), isoform CRA_b Unnamed protein product

C2 (105kDa)

C3 (70kDa)

Locus

Mass

Score

Match

Sequences

emPAI

AAC26866

87923

265

25

16

0.44

EDL31961

104971

189

14

12

0.2

EDL31961

104224

163

13

11

0.13

BAC26122

58230

95

4

3

0.06

Unnamed protein product

BAB31776

65586

75

5

3

0.05

Dhx36 protein

AAI38062

113769

74

3

3

0.03

Protease, serine, 1 precursor

NP_444473

26118

68

2

1

0.13


BAE40098

39587

42

1

1

0.08

Cask protein

AAH09740

40806

36

14

1

0.08

UbcM4-interacting protein 77

AF361001_1

23034

33

1

1

0.15

Nucleolin

AAH05460

76733

30

2

2

0.04

Nucleolin

AAH05460

76733

290

30

18

0.8

Unnamed protein product Heterogenous nuclear ribonucleoprotein U (Hnrnp U) Unnamed protein product

BAC26311

76666

290

30

18

0.42

AAC26866

87837

129

6

6

0.16

BAB31776

65586

109

8

6

0.4


BAE35231

87865

107

6

6

0.11


NP_444473

26118

107

3

3

0.18

BAA88342

62505

683

69

33

3.9

BAC37152

54882

598

58

30

3.3

NP_444473

26118

70

1

1

0.13

NP_034081

63411

62

1

1

0.05

NP_898850

65543

53

3

3

0.05

AAH47531

128708

46

6

2

0.03

AAA92993

23853

34

1

1

0.14

BAC98037

57764

33

1

1

0.06

Y box-binbing protein (YBX1)

CAA40847

35822

145

9

7

0.3


BAC26122

58230

103

4

2

0.12


BAB31776

65586

95

3

2

0.1


NP_444473

26118

93

2

1

0.27

Tcp-10

CAA41161

47082

35

1

1

0.07

AlphaCP-3

AF176327_1

35936

31

2

1

0.09

Putative hexokinase HKDC1

NP_663394

102193

30

2

2

0.03

mCG19088

EDL16344

123759

26

2

2

0.03


BAC40725

87923

190

14

10

0.25

Synaptotagmin binding, cytoplasmic RNA interacting protein (Syncrip) Unnamed protein product Protease, serine, 1 precursor Insulin-like growth factor 2 mRNA-binding protein 1 (Igf2bp1) Insulin-like growth factor 2 mRNA-binding protein 2 (Igf2bp2) Eif4g3 protein, partial Anti-diuron immunoglobulin kappa light chain, partial mKIAA0840 protein

C4 (45kDa)

N1 (135kDa)

N2 (65kDa)

N3 (33kDa)

N4 (32kDa)

Eps8l2 protein

AAH09098

44811

34

1

1

0.07

Zinc finger, HIT type 3

EDL15753

4715

33

11

1

0.79

AlphaA-CRYBP1

CAA48762

70173

31

21

1

0.05

AAH27206

60085

170

11

8

0.38

BAB31776

65586

94

2

2

0.05

Zinc finger, HIT type 3

EDL15753

4715

56

61

2

0.79

Zinc finger protein 946

NP_932120

61134

36

1

1

0.05

Cask protein

AAH09740

40806

31

33

1

0.08

AlphaA-CRYBP1

CAA48762

70173

31

62

1

0.05

AAC26867

35971

169

7

6

0.3

NP_002127

34175

56

3

3

0.1

AAH23898

114868

45

7

1

0.03

NP_001136203

96054

32

1

1

0.03

NP_002127

34175

190

8

6

0.74

BAB31776

65586

116

9

6

0.22

CAA40847

35822

110

6

5

0.56

Heterogeneous nuclear ribonucleoprotein L (Hnrnp L) Unnamed protein product

Heterogenous nuclear ribonucleoprotein A2/B1 (Hprnp A2/B1) Heterogeneous nuclear ribonucleoprotein A1 isoform a (Hnrnp A1) Eif4g3 BTB/POZ domain-containing protein KCTD1 isoform 1 Heterogeneous nuclear ribonucleoprotein A1 isoform a (Hnrnp A1) Unnamed protein product Y box-binbing protein (YBX1)

Supplementary Table 1 | MajSAT RNA binding protein candidates searched by MASCOT program. Each band detected specifically in the immunoprecipitates with MajSAT Fw probe was analyzed by LS-MS/MS. For details, refer to the user manual on website (Matrix Science, http://www.matrixscience.com/).