Supplementary Figure 1

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1 Supplementary Figure 1 Barcode analysis for the identification of mirnas that break B cell tolerance. (a) Barcode identification method. Genomic DNA samples from purified splenic B cells that escaped tolerance in the IgM b -macroself recipient mice and bone marrow B cell precursors from the same mice were subjected to a universal PCR that amplified the mirna cassette of this retroviral library. PCR products were hybridized with barcode probes coupled to specific beadsets and subsequently incubated with SA-PE. Samples were analyzed by flow cytometry for barcode identification. Adapted from reference 29. (b,c) Log2 signal distribution of a water control (b) and experimental samples (c). The background noise is represented by values lower than 7 in the x-axis. Values greater than 7 represent mirnas that were encoded by retroviruses integrated into the genomes of B cells and drove their escape from the central tolerance checkpoint imposed by the IgM b -macroself superantigen.

2 Supplementary Figure 2 No synergistic effect between mirnas that break B cell tolerance in IgM b -macroself mice. (a) Representative FACS plots showing splenic B cells (CD19 + IgM + ) in IgM b -macroself mice reconstituted with HSPCs infected with control, mir-148a or a mixture of 6 different retroviruses encoding mirnas identified in the screen (mir-148a, -26a, -26b, -342, -423, -511) at terminal analysis. (b,c) Percentages (b) and numbers (c) of splenic B cells in mice analyzed in a. Data are pooled from four individual experiments (a-c; mean s.e.m. in b-c): n=3 mice in control and retroviral mir-148a, -26a, -26b, -342, -423, -511 mixture and n=6 mice in mir-148a group.

3 Supplementary Figure 3 mir-148a expression during the development and activation of B cells. (a) Small RNA deep sequencing analysis showing mir-148a expression in murine B lineage cells at various development and activation stages. The graph was generated based on previously published data 10. (b) Taqman microrna assay showing mir-148a expression in purified B cells in the absence of stimulation or stimulated with 10 g/ml anti-igm, 10 g/ml LPS or 10 g/ml anti-igm + 10 g/ml LPS as indicated. Data are representative of two independent experiments (b; mean s.d. of technical triplicates).

4 Supplementary Figure 4 TNFR2 does not have a major role in controlling B cell central tolerance. (a) FACS analysis showing TNFR2 surface expression of non-stimulated and stimulated (2 g/ml anti- IgM for 14h) WEHI-control, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182 cells. (b) Representative FACS plots showing splenic B cells (CD19 + IgM + ) at terminal analysis of IgM b - macroself recipient mice reconstituted with bone marrow cells from mice of the indicated genotypes. (c,d) Percentages (c) and numbers (d) of splenic B cells in mice analyzed in b. Data are representative of two independent experiments (a) or pooled from two individual experiments (b-d; mean s.e.m. in c,d): n=5 mice in control and n=6 mice in Tnfrsf1b -/- group. Statistical analysis was performed with a two-tailed Student s T test. ** P<0.01.

5 Supplementary Figure 5 mir-148a, but not mir-26a or mir-182, regulates the expression of Bim, PTEN and Gadd45 simultaneously. (a) qrt-pcr analysis showing target gene mrna expression of WEHI-control, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182 stimulated with anti-igm (2 g/ml) for 14 h. (b) Western blot analysis showing mir-148a target gene expression in stimulated (2 g/ml anti-igm for 14 h) WEHIcontrol, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182. The target gene protein/ -actin ratio in the control samples was arbitrarily set as 1. Data are pooled from two individual experiments (a; mean s.d.) or pooled from two or three individual experiments for Bim and PTEN respectively (b; mean s.d.). Statistical analysis was performed with a two-tailed Student s T test. *P<0.05, ** P<0.01.

6 Supplementary Figure 6 B cell activation status and mir-148a expression in young MRL-lpr mice, and overexpression of mir-148a achieved by a retroviral vector. (a) FACS analysis showing B cell activation marker CD69 staining of splenic B cells of 6 week-old control and MRL-lpr mice. LPS-activated B cells (10 g/ml for 20h) were included as a positive control. (b) Taqman microrna assay showing mir-148a expression in splenic B cells from the control and MRL-lpr mice. Data are representative of two independent experiments (a) or pooled from two independent experiments (b; mean s.e.m.): n=3 mice/group. (c) Taqman microrna assay showing mir-148a expression in splenic B cells purified from MRL-lpr recipient mice in experiments described in Figure 6. The average mir-148a expression level of the control group was arbitrarily set as 1. Data are pooled from two independent experiments: n=6 mice in control and n=7 mice in mir-148a group. Statistical analysis was performed with a two-tailed Student s T test. ** P<0.01.

7 Supplementary Table 1. Target genes down-regulated by mir-148a in anti-igm stimulated WEHI-231 cells as identified by RNA-seq. ID Control/ mir-148a ID Control/ mir-148a ID Control/ mir-148a ID Control/ mir-148a Mafb 5.97 Arrdc Prkaa Glrx Stx Dcp Sms 1.72 Ankrd Slc2a Cnot Homer Tbc1d Csf Socs Rock Ddhd Serpine Ywhab 2.00 Rnf Lmtk Mum1l Itfg Klf Ube2w 1.55 Atp6ap Nptn 1.96 Lysmd Zc3h12c 1.55 Dll Arfip Ythdc Tnpo Tfrc 2.67 Pten 1.93 Eps Lipa 1.54 Ciita 2.62 B4galt Bcl7b 1.69 Fam178a 1.54 Cpd 2.57 Ubxn Ank Cs 1.54 Bcl2l Osbpl Kcnq Cyb5r Alcam 2.53 Kat Dmxl Gadd45a 1.53 Uhmk Usp Fam73b 1.66 Chd Fbn Pnpla Nt5c Tanc Med12l 2.39 Plekhh Fmr Gpatch Mmp Pdia Hspa4l 1.64 Arl8b 1.53 Podxl 2.39 Cul Itpk Npepl Sox5os Lrp Ppp6r Ahdc Wnt Dlgap Atxn Nceh Tmed Atp7a 1.84 Fbxo Kpna Ermp Nova Abcb Cpeb Arl6ip Epha Stard Mier Tnfrsf1b 2.23 Ptgs Epn Foxk Mex3c 2.17 Mtmr Phip 1.60 Nfat Slc45a Mospd Snx Tgoln Tpp Sgcb 1.77 Cand Tspyl Cdc14a 2.11 Btbd Hmga2-ps Nsd Nat Ammecr Cdon 1.59 Trim H2afy 2.08 Tmem9b 1.72 Dnmt

8 Supplementary Table 2. Bcl2l11, Pten and Gadd45a 3 UTRs, cloned fragments for reporter assays, and mutated mir-148a binding sites. 1. Bcl2l11 3 UTR CAGGATCTACATGCAGCCAGGATACGTGGCGGACATGGCTCTTGTTCAGACT GGGAGAACCCCCACGCGTCATGTCCCTCTCTTGGTGCTGCGACAGTGTGTCCA GTGGTTCTATCCCAGAGAGATGTGCTGAGCATGGACAGCGCTCTGCACTGTG TCGATGTGAACGGAACCTCTGTTCATCACCACATGGCCGAGTTTTCAGTAAAT ATTTGTTGTGAATGTAAACAAGGGAGGGCTTTTCTCTTTTTAATGTACAGATC CTAGGAACAGAGAAATATGCAAGAGAGGTGTTTACATGTGGCGTGTTAATTT TTTTAAACCCACTTTCTTTGATAACTGTTTTTGGGGTTTTTTTTTTTTGTCTCAT TTTCTTTCAGAAAGTAAAGCTTTTTAGCCAATTTATGGATGATTTTTATACTCA AATTCTTAACCCACTACCTACTCAGAGGATTCCATAACCCTGCAGTGGGAACT GAGCCAGCTGATTTATAAGGCTGCCTTAGTTTATTTTTAGAAATTACTGTACA GAATTTTTTAAACAGGAAGAGTATGGTGTTGCCCTCTCTCCTCTGCCATGGCC TCTTTGAGTTTCCTTTTGAGTTATTACTACCAAAACAGTTCTTAGGAAATGGA ATGAGTTCGAGCGGCAGAGGTTCTGTTCTGGTACGCCACTTCCACTTTGTAAA TATTTACCTCCTTAGCTCAGTTGCTCCTGTAGCAGAAATTCACACTTTTCCTGC TTCCCTCAACTCCTCAGACCCCTCGGTCTGTCTCCACACGGATGCGCAGCTTC AGCCCAGGGTATCTTCAAGTGTATTGTGTCAGCAAGGGCTAGGGGCTGCCCG GATTGTGCCCAGCTCCATCAATCAAGCTGACTCACGGGAAGGGCTCTCGTGC GCTTCACTGCACTGCGGTGGCTGTGTTTGCCTCTTTTATCCTGAGAGGACGTG GGCTCACACACCGCTTGCTGAAGCCGCTAGCTCCACTGGTTGCTGGCTTTGCT GCCGCCACAGGCTCTGCTTTGCTGTGTGTATCAGGTGAGCTGGAGCCCACGCT TTCTCGGGGACTGGCCAGGTCATTTCAGGAGCAAGGAGCTCAGGTGATCCTT TTCCATGCTTCTTGGGATGTAGCCCTGCTCACCCCACCTGGCCTGTGATTCCT GAATTTCATCTCTATTCCCTTGAGTTAGGGGCTGGCTCTACTTCCATCAGTTCC CAGACATAGGAAGCTTCCATTATGCCGAGTAAAGCACTTGTCTTCCACAAGA TGTCTGCCTGCTTTGCTCTCTCCATTTTCTTAATACTTGGATCTTGTTGGGCTT ACTTGTGTCTGGGGTGGGGGTGAGGTGCTCTTTCTTGGTCCCTGCCCCTTAGG ACCACAGCCCAGACATTTGGTCAGTTATTTCAAGACCAGGCAGGACTGGAAG TGCACAGCCTGGTGGAGGAGCACTTTCTAACCTGTGGAGAGCTGTGGCCCAG CTCCAGCTCCTCCCAAAACAAACGGCACACGTGTAGGCAGACAGCCAGGGG AGATACAAAAAAGGACCAAGAGGAAAGAGCATGGGAGGAGTGGGGATGGC AAGCCCTCTCACTTCTGAGAACTTCAGACGTCAGGCAGCTTGCTCAGGAAGC TTTATGAGTTTCCTCGCAAGTCAGCAGTGCCAACGACTGTTTGCTTTTGGCCT GTAGAAAGCCAGTGGTTTTTCTTATGTTATGAAGTAGAGAAATCCGTTTGTTC ATACTTTCCTCTGTGTGTGTGTGCATGTGTGTGTGTGTGTGCATATGCGTGGTT GTGTGTGTTTATATGTATATGTATATTACCTATAAGCTTCCTTCTAGATTGAGA TCATCTGATTCCAAATATCCTTATACATTTCCCAGCTTCGTTAGATTTGGTGTG GTTTGAAAGCTGTTTTGGCATTCTCCTCCTTTCCTGATGTATTGAGCCTGCTCA TGGTTCCCATGAGGCCCTGTGGTTATTATAAGTTGGTGTTAAAACTGCAACCA TGGCTCTCACACACACACACACACACACACACACACACCAAAGACATACTTG 2

9 GTCCTGGGCCATGCCCCCCTCCTAGGACCTCCATTCTCATGTGCCAGTCTCCC CGACTGGAGCACTTTACTGTCTCAGCCCCATGGCTCTGTGGCAGTGCCTGAGC AGGTGCAGAAGCTCCAGAGCCAGGTCCCAGTGACATGTGTGGCCCCCAACGC ATGCTTACCTCCAGCCTGGGGCTGGCCGCTGAAGCAGCTCATAGGGAAATAA GAGATTCTATGAATTGTAGAAGTATTCAAGAACAGGATGCGGCACCCAGAAC GTAAACATGACTGGATGTCCCTGTACTGTATCTGTTTGTTAGAGTGCAAACGT CGCCTGCAGACAGACAGTGCCCCTGCTTACTGTGGGTAGGCCTTTGTACTTAA ATATATTCTCTCTCGTTTTTGGTAGGACAGAAACTAGATCTCCCTCCCCCTCGT GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAACA TAATGCGGGTTTTGTGCCTTGACAACCTCCCCATGTGAGGTTTGTAAATAGTG TCCTGTGACTTCACCTCAAGTTGTAATAAACATACAAAATGGTGTCCTGAGTG ATTCTTCAAATTTCTTTTCCAGAAAAGAATTGATGAATTGGGGGAAGGGGCA GGGGTCTGAAAAATCAGTTTAAAGTCTCTCTATTAAAGTATGTAATGGGTAAT GTCTGCTTCTCCTATTTCCTTCCAGTGTTGTATATATTTTGACTATTTATTAGA TTAGAAAGTCATGTTTCACTCGTCAACTGAGCCAAATGTCTCTGTGCAATTGT GTTCCCTTTACCTTTCTTTGTAAAATATTGTACAGCATAAATGAGTAAAAAAA AAAAAATCACTGTTTTTCCAAACTTCTCATAATTAAGGATTTGTAAATACCAT AGGTTCTTCTTCTGTGTAATGTGCCCTACTGTTTCATAATGCTGTAACTTGTAG AGATGTTGTATATTTATTTTCCGCTTATTTAAATGTCTTAAGTTCTTGAAAGTG TTGACATCCCTATCCCTGACAGCCCTGCCATGCCATGAATTGCTTCAGCTCCT GATAAAGGAGGGGTAGTGGCTGGTTCTCTTCCCACAAGGCGGCTTCTGCTTTG GCTTCTGGCATTCAGTGCTTGGGTTCTGCCGCCTTTCTGATGAAATGAGTGTT TTGCTGCCGGTGAGATACCCTAAGATGCTAGCCTGGAAGATTGGACTCTAAG AAAATACCCATCAAACCCATTTTTGAAGCAGCTGGCTTCTGAGAAAAATCAG ACTCACCAGTAAGTTGGCCACATCTTTAGAGAAGTATTAGCAGCAGATGGGG AAGAATTTTTTTTTCCTTAAATTGAAAGAGTATCTTTGACAACTCAGTGTTGA GAGAAATATTATCCTGCTGCCTCTTGAGGTACTGCTGAATGGCTGTCCTGGGT AAGAGAAGGGCTGGTGGGACCTGTTTCTAGAGCATTTGCCAAGGCCACTTGT GTCACCAGGACACTCGCTGGCTACCGTCCCCACCTGCTGTGTGCTTCCTAGTT TGAGGTCTGTGGACTGAACTTACCTATACCATTCGGCTCCCTTCCCGTTCACT GCTTTAGGCCAGCCCACTCCTGCTTCCCTCTACAGAGGTTCTGGCACCCACGT CAAGATGGCTCACTGAAGCTCTAGAGCCATGTGACTTCATGGAAATGGTTTT GTCTTCCTGCTGATTCAGCCTGGTGCCTGTGTGTGTCCATCCGCTCAGCACGT CTGCATGCGGGTCCTGTCCACACGTGTCGCTAAATCGGCGGGGAATTGCCTCT CAAACAGCGTTGCACAAAGCACCCTCAAGTTCCCAGCAAAGTACTTGATCTC TTTCAAATGTGTGTTATTGTCTAGGTTCTGTGACCACTGGTTTCTGTACCCAGA CCTGTCAGGTATCCTTTAGTGAACAGCACTGATCTCTGTATAGTCCCCATCAC TAATGTCAGAACCCAGCGTATGTAGCATTTGTATTGCAGTTTCCCTGGCTTCC TTTACGTTTTGCACTGATGAATTTTGACAGGGTAATTGCCACTGTACCTGTGC AATACTGCTGTAAATAACTGCAGATTTTTTTAAGAAACTCAATCTTTTATGTT CTTAATGAATTTTATATAAATAAAACTTGCGACTAGCTTTCAA 3

10 (a) Bcl2l11 3 UTR fragment cloned for reporter assay Fragment Start End Bcl2l (b) mirna binding sites mirna site Position WT sequence Mutated sequence mir-148a GCACTGA GGAGTCA mir-148a TGCACTGA TCCTCAGA 2. Pten 3 UTR TTTTTTTTTTCTTATCAAGAGGGATAAAATACCATGAAAAAAAAAAAACTTGA ATAAACTGAAATGGACCTTTTTTTTTTTTTTTTTTTTTTAAATGGCAATAGGAC ATTGTGTCAGATTGCAGTTATAGGAACAATTCTCTTCTCCTGACCAATCTTGT TTTACCCTATACATCCACAGGGTTTTGACACTTGTTGTCCAGTTAAAAAAAGG TTGTGTAGCTGTGTCATGTATATACCTTTTTGTGTCAAAAGGACATTTAAAAT TCAATTAGGATAAATAAAAGATGGCACTTTCCCATTTTATTCCAGTTTTATAA AAAGTGGAGACAGGCTGATGTGTATACGCAGGAGTTTTTCCTTTATTTTCTGT CACCAGCTGAAGTGGCTGAAGAGCTCTGATTCCCGGGTTCACGTCCTACCCCT TTGCACTTGTGGCAACAGATAAGTTTGCAGTTGGCTAAGGAAGTTTCTGCAG GGTTTTGTTAGATTCTAATGCATGCACTTGGGTTGGGAATGGAGGGAATGCTC AGAAAGGAATGTTTCTACCTGGGCTCTGGACCATACACCATCTCCAGCTCCTT AGATGCACCTTTCTTTAGCATGCTCCACTTACTAATCTGGACATCCGAGAGAT TGGCTGCTGTCCTGCTGTTTGTTTGTGCATTTTAAAGAGCATATTGGTGCTAG ACAAGGCAGCTAGAGTGAGTATATTTGTAGTGGGGTACAGGAATGAACCATC TACAGCATCTTAAGAATCCACAAAGGAAGGGATATAAAAAAAGTGGTCATA GATAGATAAAAGACACAGCAGCAATGACTTAACCATACAAATGTGGAGGCTT TCAACAAAGGATGGGCTGGAAACAGAAAATTTGACAATGATTTATTCAGTAT GCTTTCTCAGTTGTAATGACTGCTCCATCTCCTATGTAATCAAGGCCAGTGCT AAGAGTCAGATGCTATTAGTCCCTACATCAGTCAACACCTTACCTTTATTTTT ATTAATTTTCAATCATATACCTACTGTGGATGCTTCATGTGCTGGCTGCCAGT TTGTTTTTCTCCTTAAATATTTTATAATTCTTCACAGGAAATTTCAACTTGAGA TTCAACAGTAAGCAGGTTTTGTTTTTTTTTTTTCCTAGAGATTGATGATGCGCG TCCTCAGTCCAGTGGCTGTCAGACGTTCAGCCCCTTTGACCTTACACATTCTA TTACAATGAGTTTTGCAGTTTTGCACATTTTTTTTAAATGTCATTAACTGTTAG GGAATTTTACTTGAATACTGAATACATATAATGTGTATATTAAAAAAGTCATT GTTTGTGTTAAAAAAGAAATTAGAGTTGCAGTAAATTTACAGCACTGCACGA ATAATAAGGCATTGAAGTTTTTCAGTAGAAATTGTCCTACAGATGCTTTATCG ACTTGCTATTGGAAGAATAGATCTTCTTAAATGTGCAGTGTTGAGTCACTTCG TTATAGTGGTAGAGTTGGGATTAGGGCTTCAATTTTACTTCTTAAATATCATT 4

11 5 CTATGTTTGATATGCCCAGACTGCATACAATTTAAAGCAAGAGTACAACTACT ATCGTAATGGTAATGTGAAGATGCTATTACAAAGGATCTCCTCCCAACCCCTC GGGAATTTGGTGTCTTTCAAATTATATCTTGACCTTGACATTTGAATATCCAG CCATTATTAGATTTCTTAATGGTGTGAAGTCCCATTTTCAATAACTTATTGGTG CTGAAATTGTTCACTAGCTGTGGTCTGACCTAGTTAATTTACAAGTACAGATT GCATAGGACCCACTAGAGAAGCATTTATAGTTTGATGGTAAGTAGATTAGGC AGAACGCCATCTAAAATATTCTTAGAAAATAATGTTGATGTATTTTCCATACC TCATCAGTTTCACTCAACCAATAAAGTTTTTAAAATTGTAACAAAGCTCTTAG GATTTACACATTTATATTTAAACATTGATACATGAATATTGACTGACTGTTGAT AAAGTCAGAGACAACTTTTCCTGAGATCTCACCATGGAAATCTGTACACCCC CTTGTCTTTCCTAAAAGCTGAAAGTGGCTGACTAAAATGCAAAGCAGCTGTT GATGTTTTGAAGATAGTGATAAACACTGTTCTTTGTTAGTTTTGGGCACAGCA TGCTAAACTATAACTTGTATTGTTCCAATATGTAACACAGAGGGCCAGGTCAT GAATAATGACATTACAATGGGCTGTTGCACTGTTAATATTTTTCCTTTGGAAT GTGAAGGTCTGAATGAGGGTTTTGATTTTGAATGTTTCAGTGTTTTTGAGAAG CCTTGCTTACATTTTATGGTGTAGTCATTGGAAATGGAAAAATGGCATTATAT ATATATTATATATATATAAATATATATATTATACATACTCTCCTTACTTTATTT CAGTTACCATCCCCATAGAATTTGACAAGAATTGCTATGACTGAAAGGGTTTT GAGTCCTAATTCAAACTTTCTTTATGACAGTATTCACGATTAGCCTGAAGTGC ATTCTGTAGGTGATCTCTCCCGTGTTTCTGGAATGCTTTCTTAGACTCTTGGAT GTGCAGCAGCTTATGTGTCTGAAATGACTTGAAGGCATCACCTTTAAGAAGG CTTACAGTTGGGCCCCGTACATCCCAAGTCCTCTGTAATTCCTCTTGGACATT TTTGCCATAATTGTAAAAGGGTAGTTGAATTAAATAGCGTCACCATTCTTTGC TGTGGCACAGGTTATAAACTTAAGTGGAGTTTACCGGCAGCATCAAATGTTTC AGCTTTAAAAATAAAAGTAGGTTACAAGTTACATGTTTAGTTTTAGAAAATTT GTGCAATATGTTCATAACGATGGCTGTGGTTGCCACAAAGTGCCTCGTTTACC TTTAAATACTGTTAATGTGTCGTGCATGCAGACGGAAGGGGTGGATCTGTGC ACTAAACGGGGGGCTTTTACTCTAGTATTCGGCAGAGTTGCCTTCTACCTGCC AGCTCAAAAGTTCGATCTGTTTTCATATAGAATATATATACTAAAACCATCCA GTCTGTAAAACAGCCTTACCCCGATTCAGCCTCTTCAGATACTCTTGTGCTGT GCAGCAGTGGCTCTGTGTGTAAATGCTATGCACTGAGGATACACAAATATGA CGTGTACAGGATAATGCCTCATACCAATCAGATGTCCATTTGTTACTGTGTTT GTTAACAACCCTTTATCTCTTAGTGTTATAAACTCCACTTAAAACTGATTAAA GTCTCATTCTTGTCATTGTGTGGGTGTTTTATTAAATGAGAGTATTTATAATTC AAATTGCTTAAATCCATTAAAATGTTCAGTAATGGGCAGCCACATATGATTAC AAAGTTCCTGTGCATTTTTCTATTTTTCCCCCTCCTTGCTATCCTTCCAAGCAAA GCATCTTTCTGTCATCTTGGTAGACACATACCTGTCTACTCATGGTTAAGAAG AGCACTTTAAGCCTTAGTCATCACTTAATAAGTTATTCCAGGCACAGTAAAAA GTTCAAGGTTCTTGGAAAACGGTGCTTATTTCTCTTCTTATAAGCCAGATGTC TGAAGATAGCCCTAACCCCAAGAACGGGCTTGATGTCTCAGGTCTGTTCTGTG GCTTTCTGTTTTTTTTAACACTGCAGTTGGCCATCAGCACATGGGAGGTTTCA TCGGGACTTGTCCAGAGTAGTAGGCTCAAATATACTATCTCCTTTCTAATATT CTTAAAGGCTAAGGAGTCCTTTCAATATAACAGTAAGATAACTTGTGATGTTT TAGAAGTAAGCAGACCATTAATGTCAATGTGGAGTCTTAATGTTACATGAAG TTGATAGTTTCTCTGTGACCCATTTAAAAATACAAACCGAGTAGCATGCAATT ATGTAAAGAAATATGAAGATTATATGTAGTCACACATTTTCTTTAGAATTCTT

12 AGTTTGGTGAAAACTTGAATATAAAGGTATTTTGATTTATATGACATTTTGAT GATATTTGAAAAAAAGGAATTTCCTGACATTTTGCTTTTAGATCATGTCCCCC ATTGTGCTGTAATTTAAGCCAACTTGGTTCAGTGAATGCCATCACCATTTCCA TTGAGAATTTAAAACTCACCAGTGTTTAACATGCAGGCTTCTGAGGGCTCCCG GAGAATCAGACCTTAAGCCCAGTTGATTTACTTCTAACGTGAAACTTCGAGTT CCTGTATACTTTGCTAGATAATTTGTGGTACATCTAAAGCTTAGTCTTAAGTG GCTTGTGTGTGGATTTTATTCAACATTCTTGTTGCTAGGGTAGAGAGAAATGT TGCTGAGTAGAAACAAGAGTACCCAGTTCAATGTGGTACAGAGAGCAGTCCC TAAAATCTGTACACAGTGTAATGGACCACTTTAGGAGTCAAGAGGCTGATTT TTCCTATGAAATTACATTGCAACAGGAAGCCTTCTAGTATAGTTCCTTTTACT GTTAGAATATGTTTTTATGCATACGCTATAGCTGCTTTCCCATCTTCCAACAA CAGGTATCAGGATGTAAGCAAGCTTTAAACAGTGTGAAGATGGCAGGATAGT GTCATCGGTAACAGTCCTCTGACTCTAAATGTAGTTGCTCTGTAACACTTTGT GAATATAACATCACAATTCTCATGTCCTTGGGGGGGGGGGGCATACCCAGTA TTAGTATGTTTTAGTGACTAAGCAATCATTTTTCTGTTTACTCATGTACATTTT CTCTTTAAAACTAAAACCTGTACTGTGTATGTCTCCAAAGCCTTTTAGCTTAG TTTTTAGGAAATGAACACTGAATGGATCACTTTTTAGTGTAGCAGGTATGGGA TATGTGCATTATAGAGAGACCTTGTCAGCTCTCTGGGCCTATTTGAATGTTTA TTGTTGGTGTGAGGATGGTAGGGGAATCAGTAAATACAAGTTACGTTGGTTT AGCAGAGCAAGCTCAGTGTGGGTATTTCTCTTTGAAGCGTGGTGCGTGACGC ACTGTGAGTAGAGAATTTGGTCACCCTTTGAGTCCTCTTGCATTTTGCAAACT TGCTCAGCAAATGCGTACCTACCTTGCCCCCTAGGTAAAAGCAGGAACTACT ACTGATTTATCTGTCACTCAGCTGTCTTTATATGTGTGCTTCTGTGACTTGTAT CACACAAGAATCTTAAAGATTTCACAAATTGTTACCTTTTAGCTCTGAATGTT GAGTATTCTGGTGGGCTAACAACAAGACAAACTCTTGACAGTCATTTGAGAA TTTTCATGAAACATTTAGCTGAAAACATTTTATAATTTATGAAAAAAATGTGT TACCTTAAACTTTTACATATGTGGGAGACATTAACTGCCATATTTGAGCATAC TGAATTTTAAATTTAAAATAAAGCTGCATATTTTTAAATGAAATGTTTAACAA GGATTCATATTTTTTGTTTTTTAAGATTAAAAATAATTTATGTCTTCTCATGTG GAACCTCATCTGTCACAATGGTTAGATTATACAGAATGGAGCAAGGCTTGTA GTGGTTTAGCTTACAGTAAAATTCTTAATGTTTAGATGTGTTTACTTACTGGCT GTTATGTATACTTTTGAGATTTTCCACCTGTTCTGTGTAGTTTTCTAAATGATA CTCCTACTTAAAAACAGCATTTTAGTATCTATTTTCTGTCTCCATTAAATGGTC CTCATTTTCTATTGAGTTTGGAAGTGTGCACATTGTGTGTGTGTGTGTGTGTGT GTGTGTGCACACGTGTGCGCGCCCGTGCGTGTGTCTATTTGTGGAGTTTGTAT GGGAGAATTAGTTTTGAAAGTGCTAGAATAGAGATGAAATTTGGTTCAAGTA AAATTTTCCCACTGGGATTTTACAGTTTATTGTAATAAAATGTTAATTTTGGA TGACCTTGAATATTAATGAATTTGTTAGCCTCTTGATGTGTGCATTAATGAGA TATATCAAAGTTGTATATTAAACCAAAGTTGGAGTTGTGGAAGTGTTTTTATG AAGTTCCGTTTGGCTACCAATGGACATAAGACTAGAAATACCTTCCTGTGGA GAATATTTTTCCTTTAAACAATTAAAAAGGTTCATTATTTTTGA 6

13 (a) Pten 3 UTR fragment cloned for reporter assay Fragment Start End Pten (b) mirna binding sites mirna site Position WT sequence Mutated sequence mir-148a TGCACTG TCCTCAG mir-148a TGCACTGA TCCTCAGA 3. Gadd45a 3 UTR TGGCATCCGAATGGAAATAACTGAACCAAATTGCACTGAAGTTTTGAAATAC CTTTGTAGTTACTCAAGCAGTCACTCCCCACGCTGATGCAAGGATTACAGAA ACTGATGTCAAGGGGCCGAGTTCAACTGCACGAGGGCTCAGAGATGACTTTG CAGAGGGAGAGAGAGGTGAGCCTGAAGAAGGAAGCTGCGAGAAAAGAGAA ATCCAAGGCAAAAGGGACAAAAACTACAAAGCACTGCAAGAAAGAAAACTG CTAATTTAGGATGGCCAGGTTACTTTCAAATAAGCCAAATATTGCTTTGTTGA AACTTTAAATGTATAGCAATAGTTTGGGTATTTTTTTTCTTTTTTTTTTTTGGTC TTTATGCCCTCAAATAAAAGGAAAGTAAAAGAGGATTAATCATATTTTCAAG CCACAGTTTAAATGTATTTTGATGAGATGTTAAATTCTCAGAAGTTTTATTAT AAATCTTACTAAGTTATTTTATGATGTGAAAGGTTATTTATGATAAAGTTTTT GAAGCACATTATCTAAAATAAACTGGTATGGAATAATTGT (a) Gadd45a 3 UTR fragment cloned for reporter assay Fragment Start End Gadd45a (b) mirna binding sites mirna site Position WT sequence Mutated sequence mir-148a TGCACTGA TCCTCAGA mir-148a GCACTG GGAGTC 7