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1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Kawai and Amano, Figure S1. Regulation of mirna expression by BRCA1. (A) Confirmation of BRCA1 overexpression. The expression level of BRCA1 was examined by qrt- PCR analysis in BRCA1-transfected or adenovirus-infected HeLa cells. (B) Expression of pri-, pre-, and mature (mat)-mirna by BRCA1 transfection in HeLa cells. (C) Expression of pri- and pre-mirna by BRCA1 transfection in HEK293 cells. (D) Expression of pri- and pre-mirna by BRCA1 transfection in MG63 cells. (E) Confirmation of BRCA1 expression after sibrca1 knockdown and in BRCA1 knockout ES cells (KO). (F) Expression of pri-, pre-, and mat-mirna by sibrca1 knockdown in HeLa cells. (G) Expression of cancer up-regulated mirna by BRCA1 overexpression in mouse BRCA1-transfected HeLa cells examined by qrt-pcr analysis. (H) Expression of non cancer-regulated mirna by BRCA1 overexpression. Error bars represent standard deviation. The broken horizontal lines represent the value of 1 used as a standard/criterion. S1
2 Figure S2. Control of mirna by BRCA1. (A) System used for in vivo monitoring of pri-mirna processing. (B) Validation of system used for in vivo monitoring of pri-mirna processing. (C) The association of BRCA1 with DROSHA was increased in a dose-dependent manner. Numbers indicate the amount of transfected DNA (in micrograms). (D) Endogenous associations of BRCA1, DROSHA, and DDX. (E) RIP analysis of BRCA1 knockdown cells. (F) RIP analysis of endogenous expression levels. Error bars represent standard deviation. The broken horizontal and vertical lines represent the value of 1 used as a standard/criterion. S2
3 Figure S3. Modulation of mirna by BRCA1 and DHX9. (A) Mouse Brca1 Ring (1 448), DBD (449 1,048), SQ (1049 1,493), and BRCT (1,494 1,811) domains were fused into the GST expression vector pgex6p1. (B) Expression of GST fusion Brca1 proteins in E. coli BL21. Arrows indicate bands of the expected proteins. (C) Human DDX5 (p68, 614 aa), DDX17 (p72, 729 aa), and DHX9 (1270 aa) were aligned, and the region around DEAD/DEIH is shown (bold). Identical amino acids are marked with asterisks, and similar amino acids with colons or periods. (D and E) Relative mrna expression of overexpressed DHX9 and sidhx9 knockdown. sidhx9 1, 9 2, and 9 3 are indicated in three sets of stealth RNAi. (F) Expression of pri-, pre-, and mat-mirna by sidhx9 knockdown. Error bars represent standard deviation. The broken horizontal lines represent the value of 1 used as a standard/criterion. S3
4 Table S1. The oligonucleotide sequences Criteria Gene Orientation Primer sequence (5-3 ) Source Cloning mp53 a Forward CACCATGACTGCCATGGAGGAGTCACAG NM_ TCATCAGTCTGAGTCAGGCCCCACTTT msmad3 a Forward CACCATGTCGTCCATCCTGCCCTTCACC NM_ CTACTAAGACACACTGGAACAGCGGAT GST-Brca1 RING Forward CGGTCGACTCATGGATTTATCTGCCGTCCAAATT NM_ GCGTCGACTTATGGTTTGGAGAAGTCTCTTCCACG GST-Brca1 DBD Forward GCGAATTCGTAGAGGATAATATCAGTGATAAA NM_ GCGTCGACTTACTTAGGCCCTCTGTTTCTACCTAG GST-Brca1 SQ Forward GCGAATTCGTGAACACTGTGCCTCCATTAGAT NM_ GCGTCGACTTACGCCTCTGATCCAGCAGGCTGGAG GST-Brca1 BRCT Forward GCGAATTCTCATCTGAGCCACACAATTCAACA NM_ GCGCGGCCGCTTAATCATTGGAGTCTTGTGGCTC sirna duplex sibrca1 Forward GGAACCUGUCUCCACAAAGdTdT Wilson and Stern, 2008 CUUUGUGGAGACAGGUUCCdTdT sidhx9-1 Forward AACAACCAAAAGCCUCCAUGGCUGCC This paper GGCAGCCAUGGAGGCUUUGGUUGUU sidhx9-2 Forward AUAAGGUUGAUACCAGCAGCUGAGG This paper CCUCAGCUGCUGGUAUCAACCUUAU sidhx9-3 Forward AAUAGCCGCCACCUCCUCUUCCCUG This paper CAGGGAAGAGGAGGUGGCGGCUAUU qrt-pcr (human) pri-let-7a-1 Forward CCTGGATGTTCTCTTCACTG Jiang et al., 2005 GCCTGGATGCAGACTTTTCT pre-let-7a-1 Forward AGGTAGTAGGTTGTATAGTTTTAGG Jiang et al., 2005 pri-mir-16-1 Forward GCAATTACAGTATTTTAAGAGATGAT Suzuki et al., 2009 CATACTCTACAGTTGTGTTTTAATGT pre-mir-16-1 Forward GCAGCACGTAAATATTGGCGT Jiang et al., 2005 CAGCAGCACAGTTAATACTGGAGA pri-mir-145 Forward TGGATTTGCCTCCTTCCCA Suzuki et al., 2009 TTGAACCCTCATCCTGTGAGCC pre-mir-145 Forward CTTGTCCTCACGGTCCAGTT This paper pri-mir-34a Forward CCTCCAAGCCAGCTCAGTTG Suzuki et al., 2009 TGACTTTGGTCCAATTCCTGTTG pre-mir-34a Forward TGGCAGTGTCTTAGCTGGTTG Jiang et al., 2005 hgapdh b Forward GTGGTCTCCTCTGACTTCAACAG NM_ CTGTAGCCAAATTCGTTGTCATAC hrnu6-1 b Forward CTCGCTTCGGCAGCACA NR_ AACGCTTCACGAATTTGCGT mbrca1 a Forward TCAAAGGACTCCTGACAACATAAA NM_ CTTTCTCAATGATTCAGTTGGATG hdhx9 b Forward TTGATCCAGTACCAGTTGGAGTAA NM_ CGTTTATGGTAATGCTTGTTTCAG pspt18 let-7a-1 Forward GCGCTAGCTCACACAGGAAACCAGGATTACCG NR_ GCGTCGACGCTGCACTACATCTCTTTAAGACA mir-16-1 Forward GCGCTAGCTGAAAAGGTGCAGGCCATATTGTG NR_ GCGTCGACTAAAAATAACAAGATTATCAATAA mir-145 Forward GCGCTAGCGCACCCCACCCTGGCTGCTACAGA NR_ GCGTCGACCTCCAGGGACAGCCTTCTTCTTGA mir-34a Forward GCGCTAGCAAGCCTGCACTACCTGCTCGCCCC NR_ GCGTCGACCTTTCCCAGCCGCCCTCACCACCC pmirglo let-7a-1 Forward GCGAATTCCTTTTCACCATTCACCCTGGATGT GCGTCGACATCAGACCGCCTGGATGCAGACTT mir-16-1 Forward GCGAATTCTCTTTTTATTCATAGCTCTTATGA GCGTCGACATATACATTAAAACACAACTGTAG mir-145 Forward GCGAATTCGGCGGCCTTGGCGCTGAAGGCCAC GCGTCGACTGGGGTGGGAAGGAGGCAAATCCA S4
5 Table S1. The oligonucleotide sequences (Continued) Criteria Gene Orientation Primer sequence (5-3 ) Source mir-34a Forward GCGAATTCCCCTCCGGATGCCGTGGACCGGCC GCGTCGACCTCGCTTCATCTTCCCTCTTGGGC RIP pri-let-7a-1 Forward CCTGGATGTTCTCTTCACTG GCCTGGATGCAGACTTTTCT pri-mir-16-1 Forward GCAATTACAGTATTTTAAGAGATGAT CATACTCTACAGTTGTGTTTTAATGT pri-mir-145 Forward TGGATTTGCCTCCTTCCCA TTGAACCCTCATCCTGTGAGCC pri-mir-34a Forward CCTCCAAGCCAGCTCAGTTG TGACTTTGGTCCAATTCCTGTTG Pull-down assay pre-let-7a-1 Forward AGGTAGTAGGTTGTATAGTTTTAGG pre-mir-16-1 Forward GCAGCACGTAAATATTGGCGT CAGCAGCACAGTTAATACTGGAGA pre-mir-145 Forward CTTGTCCTCACGGTCCAGTT pre-mir-34a Forward TGGCAGTGTCTTAGCTGGTTG EMSA c branch RNA Forward AUAGCAAUGUCAGCAGUGCCUUAGC (mir-16 based) FAM-GCUAAGGCACUGCUGACCAAAGACA linear RNA Forward UAGCAGCACGUAAAUAUUGG (mir-16 based) FAM-CCAAUAUUUACGUGCUGCUA qrt-pcr (mouse) pri-let-7a-1 Forward CCTGGATGTTCTCTTCACTG This paper CCATGAATGCAGACTTTTCC pre-let-7a-1 Forward AGGTAGTAGGTTGTATAGTTTTAGG Jiang et al., 2005 pri-mir-16-1 Forward TGGTAATGCAGCTCAGTTGG This paper GACTTGCTTTATGGCCAAGG pre-mir-16-1 Forward GCAGCACGTAAATATTGGCGT This paper CAGCAGCACAGTCAATACTGGAGG pri-mir-145 Forward TTGGGAGCTCTCTTCTGACC This paper CTCCCTCCTGGGGATGTTAG pre-mir-145 Forward CTTGTCCTCACGGTCCAGTT This paper pri-mir-34a Forward AAGGTCAGAGGTCAGCAACG This paper CCAGAAGTGCTCACACTCCA pre-mir-34a Forward TGGCAGTGTCTTAGCTGGTTG Jiang et al., 2005 Underlines show matched complementary sequence. These oligonucleotides were partially annealed and used for the experiment. a mp53, msmad3, and mbrca1 represent mouse homologue of p53, Smad3, and Brca1. b hgapdh, hrnu6-1, and hdhx9 represent human homologue of GAPDH, RNU6-1, and DHX9. c These oligonucleotides were synthesized based on mir-16. References Jiang, J., E.J. Lee, Y. Gusev, and T.D. Schmittgen Real-time expression profiling of microrna precursors in human cancer cell lines. Nucleic Acids Res. 33: Suzuki, H.I., K. Yamagata, K. Sugimoto, T. Iwamoto, S. Kato, and K. Miyazono Modulation of microrna processing by p53. Nature. 460: dx.doi.org/ /nature08199 Wilson, K.A., and D.F. Stern NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway. Cell Cycle. 7: dx.doi.org/ /cc S5
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doi:.38/nature899 Supplementary Figure Suzuki et al. a c p7 -/- / WT ratio (+)/(-) p7 -/- / WT ratio Log X 3. Fold change by treatment ( (+)/(-)) Log X.5 3-3. -. b Fold change by treatment ( (+)/(-)) 8
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A. TCTAGTTGTTGTTGTTATGGTCTAGTTGTTGTTGTTATGGTCTAATTT AAATATGGTCTAAAGAAGAAGAATATGGTCTAAAGAAGAAGAATATGG 2XP35S STTM165 5 GGGGGATGAAGctaCCTGGTCCGA3 3 CCCCCUACUUC---GGACCAGGCU5 mir165 HindIII mir165 96 nt GTTGTTGTTGTTATGGTCTAGTTGTTGTTGTTATGGTCTAATTT
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Materials and Methods Transgenic Plant Materials and DNA Constructs. VEX1::H2B-GFP, ACA3::H2B-GFP, KRP6::H2B-GFP, KRP6::mock21ts-GFP, KRP6::TE21ts- GFP and KRP6::miR161ts-GFP constructs were generated