Supporting Information
Tan and Elowitz 10.1073/pnas.1320873111
TTP
Brf1
Brf2
Hours
0
1.0
1.5
2.0
2.5
0.25
1.00
0.50
2.00
Fold Change in mRNA
mRNA Expression aer
MEK1/2 inhibion (CI-1040)
Brf1
GAPDH
0
1.0
1.5
2.0
Hours
(1.00)
(0.80)
(0.51)
(0.66)
(1.00)
(0.84)
(0.67)
(0.85)
TTP
(1.00)
(1.06)
(0.99)
(1.17)
Brf2
Protein Expression aer
MEK1/2 inhi
bion (CI-1040)
0
0.25
0.5
1
2.5
5
7.5
15
25
Erk1/2
pp-Erk1/2
E14 mESCs (+ CI-1040, MEK inhibitor)
Fold Change
pp-Erk1/2
Hours
A
C
B
D
α
-Brf1
α
-Brf2
α
-TTP
α
-GAPDH
Fig. S1.
(
A
) A 2.5-h quantitative RT-PCR time course of TTP, Brf1, and Brf2 mRNA level changes in response to MEK1/2 pharmacological inhibitors (
±
SEM;
n
=
3
for
t
≤
2h;
n
=
2for
t
=
2.5 h). (
B
) Representative Western blots of TTP, Brf1, and Brf2 protein levels for a 2.5-h time course. (
C
) Pharmacological inhibition of
MEK1/2 with 5
μ
M PD184352/CI-1040 inhibits Erk1/2 activation for
>
24 h. (
D
) Zfp36 AU-rich element mRNA-binding proteins (AUBPs) contain AU-rich elements
(AREs) within their own 3
′
-UTRs and have the capacity to directly autoregulate and cross-regulate their own expression. siRNA knockdown of individual Zfp36
AUBPs (10 nM final siRNA concentration) leads to corresponding protein level changes in the remaining Zfp36 AUBPs. Protein levels were assayed 36 h af
ter
siRNA transfection by Western blot.
Tan and Elowitz
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IL-2 mRNA and ARE Clusters
3’UTR
AAAAAAA
m
7
G
1
2
IL-2 CDS
A
C
0.25
0.50
1.00
Fold Change in Brf1 mRNA
Wildtype E14 mESCs
Wildtype E14 mESCs
+ FGFR inhibitor (5 hours)
Wildtype E14 mESCs
+ MEK1/2 inhibitor (5 hours)
Fgf4-
/
-
R1 mESCs
Fgf4
-/
-
R1 mESCs
+ FGF4/Heparin (5 hours)
Relave Differences in Brf1
between Cell Types and Condions
B
Androgen Receptor (AR) 3’UTR sequence:
polyA sequence:
5’
(AAAAAAAAAA)
12
3’
5’
CUGGGCUUUUUUUUUCUCUUUCUCUCCUUUCUUUUUCUUCUUCCCUCCCUA
3’
Bead
5’
Nanog RNA
ARE 1
ARE 2&3
ARE sites 1, 2 and 3
wildtype
ARE site 1
Input lysate
No RNA
Nanog RNA
variants
Nanog RNA +
(AR) sequence
Nanog RNA +
polyA sequence
PCBP1
PCBP1 binding site
Fig. S2.
(
A
) Cartoon depicting the approximate location and sequence of AREs within the 3
′
-UTR of IL-2, used as a control in the protein pull-down assay. (
B
)A
Western blot for poly(C) RNA-binding protein 1 (PCBP1) was performed as an RBP recovery control. We appended an androgen receptor (AR) 3
′
-UTR sequence
that is known to bind PCBP1 (site underlined) to the 3
′
end of wild type and ARE mutant versions of Nanog (cartoon). The presence or absence of ARE se-
quences does not inhibit the binding of PCBP1 to its target sequence. Moreover, the absence of PCBP1 binding sequence prevents association with PCBP1
[tested with a poly(A) sequence of 120 nt]. (
C
) Relative differences in Brf1 expression level among different cell types and cell culture conditions, as specified.
Table S1. Frequency of different ARE motifs among the 18,313
protein coding genes in the NCBI37/mm9 mouse transcript
annotation
Motif
% of annotation
Consensus
-
ATTTATTTATTTATTTATTTA
-
0.08
Full
-
ATTTATTTATTTATTTA
-
0.2
Full
-
ATTTATTTATTTA
-
0.5
Full
-
ATTTATTTA
-
4.5
Full
-
TTATTTATT
-
6.0
Full (minimal)
-
TATTTAT
-
22.8
Partial (minimal)
-
CTATTTATT
-
2.2
Partial
-
TTATTTATC
-
1.0
Partial
-
CTATTTATC
-
0.5
Partial
Tan and Elowitz
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Table S2. Primer and probes characterization for qPCR
Gene name
Accession no.
Primers and probe
Slope/efficiency/
R
2
Citrine (YFP)
Primer1: 5
′
-
CCACCTTCGGCTACGGCCTGA
-3
′
3.24/116%/0.998
Primer2: 5
′
-
GCCATGATATAGACGTTGTGG
-3
′
Dab2
NM_023118.5 Primer1: 5
′
-
TGTACTTTGTGGGTTCTGTCC
-3
′
3.59/90%/0.999
Primer2: 5
′
-
GGTTGTGCTTGTTTCTACTTCG
-3
′
Probe: 5
′
-
CTGCTTGCCTTCCCGTCATGTCTAA
-3
′
Esrr
β
NM_011934.4 Primer1: 5
′
-
CCTTTACTATCTGTGCCTGGTC
-3
′
3.27/102%/0.998
Primer2: 5
′
-
AGTGCTTCTCTTTGGTGCTG
-3
′
Probe: 5
′
-
ACACGTCTGTCATCCTTGCCTGC
-3
′
Fgf5
NM_010203.4 Primer1: 5
′
-
TGACTGGAATGAGTGCATCTG
-3
′
3.40/97%/1.000
Primer2: 5
′
-
GGGTTTGGAATTTGGGTTGAG
-3
′
Probe: 5
′
-
ATTAAGCTCCTGGGTCGCAAGGG
-3
′
FoxA2
NM_010446.2 Primer1: 5
′
-
GATGTACGAGTAGGGAGGTTTG
-3
′
3.21/105%/0.999
Primer2: 5
′
-
AACATGAACTCGATGAGCCC
-3
′
Probe: 5
′
-
CCAAGACATACCGACGCAGCTACA
-3
′
Gapdh
NM_008084.2 Primer1: 5
′
-
CTCCACGACATACTCAGCAC
-3
′
3.38/97%/0.999
Primer2: 5
′
-
CCACTCACGGCAAATTCAAC
-3
′
Probe: 5
′
-
AGGAGCGAGACCCCACTAACATCA
-3
′
Gata6
NM_010258.3 Primer1: 5
′
-
AGCAAGATGAATGGCCTCAG
-3
′
3.35/99%/1.000
Primer2: 5
′
-
CTCACCCTCAGCATTTCTACG
-3
′
Probe: 5
′
-
CAACTGTCACACCACAACCACTACCT
-3
′
Gbx2
NM_010262.3 Primer1: 5
′
-
GCAGTGTTTTGAAAGGGATAGG
-3
′
3.63/89%/0.999
Primer2: 5
′
-
TGTTTGTCCTTGTGTCTCCTG
-3
′
Probe: 5
′
-
TTTGGGCACGTATGGGAAGGTGG
-3
′
Hnf4
α
NM_008261.2 Primer1: 5
′
-
GGGCAGGAGAAGGATAAGAAAG
-3
′
3.68/87%/0.996
Primer2: 5
′
-
GCAAAGCCATCAAGAGTCAAC
-3
′
Probe: 5
′
-
TGGGAAGCTACAGTCAAGGTGCATT
-3
′
Hprt
NM_013556.2 Primer1: 5
′
-
GCCCCAAAATGGTTAAGGTTG
-3
′
3.36/98%/0.999
Primer2: 5
′
-
AACAAAGTCTGGCCTGTATCC
-3
′
Probe: 5
′
-
CTTGCTGGTGAAAAggacctctcgaa
-3
′
Nanog
NM_028016.2 Primer1: 5
′
-
CAAAGGATGAAGTGCAAGCG
-3
′
3.35/99%/0.999
Primer2: 5
′
-
CCAGATGCGTTCACCAGATAG
-3
′
Probe: 5
′
-
CAGCACCAGTGGAGTATCCCAGC
-3
′
Nodal
NM_013611.4 Primer1: 5
′
-
TTCACCGTCATTCCTTCTCAG
-3
′
3.38/98%/1.000
Primer2: 5
′
-
GATGCCAACACTTTTCTGCTC
-3
′
Probe: 5
′
-
ACACCTGCTTTTCCAGTGCCCT
-3
′
Oct4
NM_013633.3 Primer1: 5
′
-
CACTCTACTCAGTCCCTTTTCC
-3
′
3.34/99%/1.000
Primer2: 5
′
-
GTTCTCTTGTCTACCTCCCTTG
-3
′
Probe: 5
′
-
TTTCCCTCTGTTCCCGTCACTGC
-3
′
Rex1
NM_009556.3 Primer1: 5
′
-
ACATCCTAACCCACGCAAAG
-3
′
3.17/107%/1.000
Primer2: 5
′
-
CATTAAGACTACCCAGCCTGAG
-3
′
Probe: 5
′
-
TGTCTCCACCTTCAGCATTTCTTCCC
-3
′
Sdha
NM_023281.1 Primer1: 5
′
-
AGTGGGCTGTCTTCCTTAAC
-3
′
3.21/105%/1.000
Primer2: 5
′
-
GGATTGCTTCTGTTTGCTTGG
-3
′
Probe: 5
′
-
TGGGCATGTCTCTGAGGGATTGG
-3
′
Sox1
NM_009233.3 Primer1: 5
′
-
TCTTTCCTGTGGTTCTGCC
-3
′
3.79/84%/0.999
Primer2: 5
′
-
GAAATCAAAGGCACGCTGTC
-3
′
Probe: 5
′
-
TTGTCCCTATCCTTGGCCTTGTCC
-3
′
Sox2
NM_011443.3 Primer1: 5
′
-
CCAATCCCATCCAAATTAACGC
-3
′
3.78/84%/0.999
Primer2: 5
′
-
CTATACATGGTCCGATTCCCC
-3
′
Probe: 5
′
-
CCGCCCTCAGGTTTTCTCTGTACAA
-3
′
T
NM_009309.2 Primer1: 5
′
-
GCTGGAAATATGTGAACGGG
-3
′
3.87/81%/1.000
Primer2: 5
′
-
GTTGGTGAGTTTGACTTTGCTG
-3
′
Probe: 5
′
-
AAAATTGGGCGAGTCTGGGTGGA
-3
′
Tbp
NM_013684.3 Primer1: 5
′
-
TGATTGCTGTACTGAGGCTG
-3
′
3.24/104%/0.998
Primer2: 5
′
-
CTTACGGCACAGGACTTACTC
-3
′
Probe: 5
′
-
ACTGTTGGTGTTCTGAATAGGCTGTGG
-3
′
Tbx3
NM_011535.2 Primer1: 5
′
-
TCCCATTATCCTCAACCTTGC
-3
′
3.48/94%/0.998
Primer2: 5
′
-
CACACGAAGCCCTCTACAAG
-3
′
Probe: 5
′
-
CCATAGCTGCCCCTTTTACCCCA
-3
′
Zfp36 (TTP)
NM_011756.4 Primer1: 5
′
-
CCCTGTCCTCTTGTTCCTTTTC
-3
′
3.14/108%/1.000
Primer2: 5
′
-
TGGTTAGGGTCTCTTCGAGTC
-3
′
Probe: 5
′
-
CTTTCCCCTTCTGCCTTCTCTGCT
-3
′
Tan and Elowitz
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Table S2. Cont.
Gene name
Accession no.
Primers and probe
Slope/efficiency/
R
2
Zfp36l1 (Brf1) NM_007564.5 Primer1: 5
′
-
GCACACTCTTCCTCTTCCTTATAG
-3
′
3.23/104%/0.997
Primer2: 5
′
-
AGGCAAGATTAGTCAACAGGG
-3
′
Probe: 5
′
-
ACCTTTTACTTCCCAGCCCGAACC
-3
′
Zfp36l2 (Brf2) NM_001001806.2 Primer1: 5
′
-
TCGCCCGTTATTCATCTTGG
-3
′
3.17/107%/1.000
Primer2: 5
′
-
CGTAGAAGGGTGACAGAAGTG
-3
′
Probe: 5
′
-
AAGCGTGGAGGTTGGGAGGT
-3
′
1/2 life tag
Primer1: 5
′
-
CTGTTTTGACCTCCATAGAAGAC
-3
′
3.32/100%/0.999
Primer2: 5
′
-
AGATTGAGGATGCTGAGCGTC
-3
′
Dataset S1. Transcript abundances in fragments per kilobase exon per million mapped reads (FPKM) from two biological replicates of
three conditions [total RNA, Brf1/2 antibody RNA/RBP immunoprecipitation (RIP), and IgG control antibody RIP]
Dataset S1
Dataset S2. Values used to compute
E
RIP
enrichment statistic
Dataset S2
Dataset S3. Identifying the presence (
“
1
”
) or absence (
“
0
”
) of specific ARE motifs in the 3
′
-UTR of all protein coding genes
Dataset S3
Absence of any value indicates no 3
′
-UTR was found in the annotation.
Dataset S4. Documenting the location of ARE motifs in the 3
′
-UTR of all protein coding genes
Dataset S4
Numerical values indicate the starting position (5
′
end) of the ARE motif relative to the first 3
′
-UTR nucleotide. Absence of any value indicates no ARE motif
is present.
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