The Targeting and Functions of miRNA-383 are Mediated by FMRP during Spermatogenesis
Supplementary Information
Supplementary Figures
Supplementary Figure S1. Primary miR-383 expression was downregulated in testes of FMRP KO mice (A), while a loss of FMRP did not affect the levels of primary miR-383 in NT2 cells (B). After 50 nM siRNA of FMRP was transfected into NT2 cells for 48 h, the pri-miR-383 expression was measured using real-time PCR.
Supplementary Figure S2. FMRP was downregulated by miR-383, but not a target of miR-383. (a and b) Expression of FMRP was reduced by miR-383 overexpression in Hela (panel a) and NIH3T3 (panel b) cell lines, respectively. Cells were harvested 48 h after transfection of mimic NC or miR-383 mimic for Western blot analysis. (c) miR-383 did not target the 3′ UTR and CDS region of FMRP. Luciferase reporters containing the FMRP 3' UTR or CDS region were cotransfected with either mimic NC or miR-383 mimic into HEK293T cells. Luciferase activity was measured 30 h after transfection. Data are presented as means ± SEM from three experiments performed in triplicate.
Supplementary Figure S3. Alteration of pRb phosphorylation by RA or serum stimulation can regulate FMRP protein levels. (a and b) Different concentrations of RA were added to NT2 (panel a) and GC1 (panel b) cell cultures for 24 h, and the level FMRP was measured by Western blot analysis. (c and d) Serum stimulation upregulated FMRP expression in NT2 (panel c) and GC1 (panel d) cells in a time-dependent manner. Cells were starved with serum free medium for 24 h and stimulated with serum for 0, 2, 4, 8, 12, 16 and 24 h, and then proteins were extracted for Western blot detection.
Supplementary Figure S4. γH2AX was detected in nuclear spreads of Fmr1 WT and KO pachytene spermatocytes collected from adult mice. Arrow indicated the γH2AX signal on the autosomes Red, SCP3; green, γH2AX. There is no significant alteration of MLH1 foci on synapsed chromatin in FMRP KO meiocytes. MLH1 was detected in nuclear spreads of FMRP WT and KO pachytene spermatocytes collected from adult mice. Red, SCP3; green, MLH1. Scale bar: 20 μm.
Supplementary Tables
Supplementary Table 1. Altered FMRP associated miRNAs in testes of NOA patients according to small RNA deep sequencing results.
Compared with normal controlsSpermatogonia arresst / Spermatocyte arrest / Hypospermatogenesis
let-7d-3p / ↓ / ↓ / ↓
miR-101b-3p / ↓ / - / ↓
miR-106b-5p / ↑ / - / ↑
miR-10a-3p / ↓ / - / ↓
miR-129-2-3p / ↑ / ↑ / ↑
miR-132-5p / ↓ / ↓ / -
miR-145-5p / ↑ / ↑ / -
miR-149-3p / ↑ / - / ↑
miR-17-3p / - / ↑ / -
miR-185-5p / ↓ / ↓ / -
miR-204-3p / - / ↑ / -
miR-20a-5p / ↑ / ↑ / ↑
miR-210-3p / ↑ / ↑ / ↑
miR-214-3p / - / ↓ / ↑
miR-22-5p / ↑ / ↑ / -
miR-24-1-5p / ↑ / ↑ / ↑
miR-24-2-5p / ↑ / ↑ / -
miR-26b-5p / - / ↓ / ↓
miR-29b-1-5p / ↓ / - / -
miR-29c-3p / ↓ / - / -
miR-30c-1-3p / ↓ / ↓ / ↓
miR-320-3p / ↓ / ↑ / ↓
miR-329-3p / ↑ / - / -
miR-331-3p / ↑ / ↑ / ↑
miR-335-5p / ↓ / ↓ / -
miR-34b-5p / ↓ / - / ↑
miR-383-5p / ↓ / ↓ / ↓
miR-425-3p / ↑ / ↑ / ↑
miR-450a-1-3p / - / - / ↓
miR-450a-2-3p / ↑ / - / ↓
miR-455-5p / ↑ / ↑ / -
miR-485-3p / ↑ / - / -
miR-505-5p / ↑ / ↑ / ↑
miR-532-5p / - / ↑ / -
miR-665-3p / ↑ / - / -
miR-671-3p / ↑ / ↑ / ↑
miR-744-5p / ↑ / ↑ / -
Supplementary Table S2. List of primer pairs for the construction of vectors.
Cyclin D1-3'UTR WT* / Forward: GCACGCTCGAGTGTTGAAGGGAGGTGGCAAGAG
Reverse: GCATTGCGGCCGCAGGATGGTTGAGGTAAGCGTGA
Cyclin D1-3'UTR MT** / Forward: TCATCTGTAGGGGGGCGTAGCATCATAGTAG
Reverse: CGCCCCCCTACAGATGAAGTGCCCAGCATCA
FMRP-3'UTR 1 / Forward: CGTCTCGAGCTGCATAATTCTGAAGTTATATTTC
Reverse: GCAGCGGCCGCAGTGGTACCCCTACTAGGAGTT
FMRP-3'UTR 2 / Forward: CGTGCCTCGAGCCACTGAATCTGTACAGAGCCGTA
Reverse: GCATTGCGGCCGCGAATGAACTCCAACTCCATTTG
FMRP-CDS 1 / Forward: CGTGCGATCGCGAAGAGAAGATGGAGGAGCTGGT
Reverse: GCATTGCGGCCGCGCGATGCTGTCCTTTGTTCCC
FMRP-CDS 2 / Forward: CGTCTCGAGCTAATGCCACTGTTCTTTTGG
Reverse: GCAGCGGCCGCTTTAGGGTACTCCATTCACGA
Supplementary Table S3. Sequence of oligonucleotides.
Inhibitor NC / CAGUACUUUUGUGUAGUACAA
miR-383 inhibitor / AGCCACAAUCACCUUCUGAUCU
mimic NC & si.NC / UUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATT
miR-383 mimic / AGAUCAGAAGGUGAUUGUGGCU
CCACAAUCACCUUCUGAUCUUU
LNA scramble-miR / GTGTAACACGTCTATACGCCCA
LNA hsa-miR-383 / AGCCACAATCACCTTCTGATCT
LNA mmu-miR-383 / AGCCACAGTCACCTTCTGATCT
Supplementary Table S4. List of primer pairs for real-time PCR.
Gene / Sequence (5' to 3') / Annealing temperature (°C)
β-actin (human) / Forward: TGGCACCCAGCACAATGAA / 60
Reward: CTAAGTCATAGTCCGCCTAGAAGCA / 60
β-actin (mouse) / Forward: TGGCTCCTAGCACCATGAA / 60
Reward: CTCAGTAACAGTCCGCCTAGAAGCA / 60
PGK1 (human) / Forward: TAAAGGGAAGCGGGTCGTTATG / 60
Reward: ACAGCAACTGGCTCTAAGGAGTACTTG / 60
PGK1 (mouse) / Forward: GAAGGGGAAGCGGGTCGTGATG / 60
Reward: GCAGCAACTGGCTCTAAGGAGTACTTG / 60
FMRP (human) / Forward: CACCGCTCCAACAGAGGAAGAGAG / 60
Reward: TTAGGGTACTCCATTCACGAGTG / 60
FMRP (mouse) / Forward: CCGAACAGATAATCGTCCACG / 60
Reward: ACGCTGTCTGGCTTTTCCTTC / 60
IRF-1 (human) / Forward: CGAAGTCCAGCCGAGATGCTA / 60
Reward: TGCTGTGGTCAGGCAGAG / 60
IRF-1 (mouse) / Forward: GGGAAGATAGCCGAAGACCT / 60
Reward: CCTCGAGGGCTGTCAATCT / 60
Cyclin D1 (human) / Forward: ACAAACAGATCATCCGCAAACAC / 60
Reward: TGTTGGGGCTCCTCAGGTTC / 60
Cyclin D1 (mouse) / Forward: ACAAGCAGACCATCCGCAAGCAT / 60
Reward: TGTTGGGGCTGCCCAGGTTC / 60
CDK4 (human) / Forward: TCGAAAGCCTCTCTTCTGTG / 60
Reward: TACATCTCGAGGCCAGTCAT / 60
CDK4 (mouse) / Forward: AATGTTGTACGGCTGATGGA / 60
Reward: AGAAACTGACGCATTAGATCCT / 60
Dicer (human) / Forward: GTACGACTACCACAAGTACTTC / 60
Reward: ATAGTACACCTGCCAGACTGT / 60
Dicer (mouse) / Forward: GTATGACTACCACAAGTACTTT / 60
Reward: ATAGTACACCTGCCAGACCAC / 60
Drosha (human) / Forward: GTGCTGTCCATGCACCAGATT / 60
Reward: TGCATAACTCAACTGTGCAGG / 60
AGO1 (human) / Forward: TGGTGCTCTCCCTTACTCTAC / 60
Reward: CTCCCTTTCCCACCTCATTCTG / 60
AGO2 (human) / Forward: CCAGCTACACTCAGACCAACAGA / 60
Reward: GAAAACGGAGAATCTAATAAAATCAATGAC / 60
MAP1B (human and mouse) / Forward: AAGCTGAGAGGTCCCTTATGTCA / 60
Reward: GATTAGCTCCAGCTGAGGCTTG / 60
pri-miR-383 / Forward: CTTTCCCAAGAGTTTCACT / 60
Reward: CCACTCCAGTCCACCAAAT / 60
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