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Supplemental Inventory
Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expressionShihoko Kojima, Elaine L. Sher-Chen, Carla B. Green
Main Supplemental File includes:
8 Supplemental Figures
Supplemental Table S3; PAT assay
Supplemental Table S1; RESS mRNAs
Supplemental Table S2; PAR mRNAs
Supplemental Table S4; Oligos
Kojima_Supplemental Fig. 1
Gen
es
ZT 0 12 24 12 24
ZT 12
ZT 0
ZT 4
ZT 8ZT 16
ZT 20
A B
Supplemental Fig. 1. RESS mRNAs have steady-state mRNA
rhythms with peak phases distributed around the clock. A) Phase-
sorted heatmap of steady-state expression level of all RESS
mRNAs in mouse liver from our microarray analysis. Red color
indicates higher expression and green color represents lower
expression based on microarray analyses. Each line represents one
mRNA. Heat-map is double-plotted for easier visualization of
rhythmicity. B) Peak phase distribution of RESS mRNAs. Peak time
of RESS mRNAs was calculated based on the expression level from
microarray and each segment represents a 2-hr block.
Kojima_Supplemental Fig. 2
Log
10
(Lon
g/Sh
ort)A B C D
Log2(Poly(A)+)Log2(Short)Log2(Poly(A)+)
ZT
4Z
T8
ZT
12Z
T16
ZT
20
Log
10
(Lon
g/Sh
ort)
Log
10
(Lon
g/Sh
ort)
Log
10
(Lon
g/Sh
ort)
Log
10
(Lon
g/Sh
ort)
Log 2
(Lon
g)
y=0.9934xR2=0.9707
Log 2
(Lon
g)
y=1.0004xR2=0.9757
Log 2
(Lon
g)
y=1.0009xR2=0.9769
Log 2
(Lon
g)
y=1.0181xR2=0.9764
Log 2
(Lon
g)
y=1.0110xR2=0.9781
Log 2
(Sho
rt) y=0.9873x
R2=0.9884
Log 2
(Sho
rt) y=0.9927x
R2=0.9914
Log 2
(Sho
rt) y=1.0083x
R2=0.9880
Log 2
(Sho
rt) y=0.9795x
R2=0.9906
Log 2
(Sho
rt) y=0.9882x
R2=0.9921
Log 2
(Lon
g)
y=0.9939xR2=0.9926
Log 2
(Lon
g)y=1.0027xR2=0.9914
Log 2
(Lon
g)
y=1.0195xR2=0.9902
Log 2
(Lon
g)
y=1.0084xR2=0.9941
Log 2
(Lon
g)
y=1.0062xR2=0.9877
Log2(Poly(A)+)
Supplemental Fig. 2. Comparison of the expression levels of the various fractions from the
microarray data set for each time point (except for ZT 0 which is shown in Figure 1). Shown
is the comparison of the expression level for each mRNA between the polyA+ samples
versus the short poly(A) tail samples (A), between the polyA+ samples versus the long tailed
samples (B), between the short tailed samples versus the long tailed samples (C), and
between the total polyA+ samples versus the log10(long/short ratio) (D). The degree of
correlation is also shown in the upper left hand corner of each graph.
Poly(A
) length (nt)Rel
ativ
e ex
pres
ion
Poly(A) tail length (LM-PAT)
Pre-RNA
Steady-state RNA (qPCR)
0
60
120
0
0.8
1.6
60
100
140
0
3
6
0
60
120
0
2
4
Rbm3
Aqp8
Abhd14a
A Class I; Pre-RNA; Rhythmic, Steady-state; Rhythmic, Poly(A) tail; Rhythmic
B Class II; Pre-RNA; Rhythmic, Steady-state; Non-Rhythmic, Poly(A) tail; Rhythmic
C Class III, Pre-RNA; Non-Rhythmic, Steady-state; Non-Rhythmic, Poly(A) tail; Rhythmic
0
60
120
0
0.8
1.6
100
150
200
0
1.2
2.4 C2
Fbxo36
-40
0
40
0
1
2Asl P
oly(A) length (nt)
Rel
ativ
e ex
pres
sion
Poly(A
) length (nt)
Rel
ativ
e ex
pres
sion
Rdh9
Gstm3
0
70
140
0
1
2
60
120
180
0
2.5
5
ZT 0 12 24 12 24 0 12 24 12 24 0 12 24 12 24
ZT 0 12 24 12 24 0 12 24 12 24 0 12 24 12 24
ZT 0 12 24 12 24 0 12 24 12 24
Kojima_Supplemental Fig. 3
Supplemental Fig. 3. Additional examples of the expression patterns of PAR mRNAs from each class. A) Class I
mRNAs; Asl (left), Aqp8 (middle), and Rdh9 (right), B) Class II mRNAs; C2 (left), Abhd14a (middle), and Gstm3
(right), C) Class III mRNAs; Fbxo36 (left) and Rbm3 (right). All the rhythmicity was assessed by Circwave v1.4.
The mean pre-mRNA (green) and the steady-state mRNA levels (blue) (n=3 for each time point) were measured
by qPCR. Poly(A) tail length (orange) was calculated from LM-PAT assays of pooled samples (n=3 for each time
point). All the graphs are double-plotted for easier visualization of rhythmicity.
A50
A100
A0
Aqp8Abhd14a
A100
A200
A0
Asl
A100
A200
A0
Cyp2a4
A100
A200
A0
Gstm3
A100
A200
A0
Hsd17b6
A100
A200
A0
Rbm3
A100
A200
A0
Rdh9
A100
A200
A0
Slc22a1
A100
A200
Kojima_Supplemental Fig. 4
Supplemental Fig. 4. Additional gel images of LM-PAT assay for PAR mRNAs.
Each lane consists of pooled samples (n=3 for each time point). Arrowheads on
the right indicate the fragment derived from restriction enzyme treatment for
verification of PCR specificity (see Materials and Methods for details ), while
arrows represent non-specific amplification.
A B
ZT 12
ZT 0
ZT 4
ZT 8ZT 16
ZT 20
ZT 12
ZT 0
ZT 4
ZT 8ZT 16
ZT 20
Pea
k tim
e di
ffere
nce
(hrs
)(S
tead
y-S
tate
) –
(Pre
-RN
A)
RESS(non-PAR)
0
1
2
3
4
5
6
PARClass I
***C
% G
ene
num
ber
Peak time difference (hrs)
0
5
10
15
20
-12 -9 -6 -3 0 3 6 9 12
Long/short ratio peak D
Kojima_Supplemental Fig. 5
Supplemental Fig. 5. Peak distribution of the poly(A) tail length (A), and steady-state
mRNA expression (B) of the entire set of Class I PAR mRNAs from microarray. C)
Distribution of peak time differences of steady-state mRNA expression from long/short ratio
peak time of Class I mRNAs. D) Average peak time difference between pre-mRNA and
steady-state mRNA expression of Class I PAR and RESS (excluding PARs) mRNAs (means
± SEM). ***; p<0.0001 (Student’s t-test).
Cpeb1C
peb1
/36B
4Cpeb2
Cpe
b2/3
6B4
0
0.5
1
1.5
0
0.5
1
1.5
Kojima_Supplemental Fig. 6
Supplemental Fig. 6. The mRNA expression of Cpeb1 (left) or Cpeb2 (right)
upon knocking-down by siRNAs in AML12 cells. The level of control siRNA
was set as 1.
Enzyme(-) Enzyme(+)
Mif
Gstt2
Hspb1Hsp27
Pdzk1ip1
Slc44a3
Kojima_Supplemental Fig. 7
Supplemental Fig. 7. Gel images of LM-PAT assay with or without restriction
enzyme treatment for PAR mRNAs shown in Figure 5. Each lane consists of pooled
samples (n=3 for each time point). Arrowheads on the right side indicate the
fragment derived from restriction enzyme treatment for verification of PCR
specificity (see Materials and Methods for details). This could not be visualized for
Slc44a3 because of the size of excised band (51 bp), although the all the bands in
the left panel was shifted downward with enzyme treatment.
0 12 24 12 240
0.4
0.8
0
0.3
0.6
0 12 24 12 240
1
2
0 12 24 12 24
0
0.7
1.4
0 12 24 12 24 0 12 24 12 240
0.7
1.4
0
0.8
1.6
0 12 24 12 24
0
0.5
1
0 12 24 12 24ZT
0
0.4
0.8
0 12 24 12 24
0
0.8
1.6
0 12 24 12 24
0
0.2
0.4
0 12 24 12 24
Noc/Ccrn4lCcr4b/Cnot6lCcr4a/Cnot6 Ccr4d/Angel1 Ccr4e/Angel2
Caf1a/Cnot7 Caf1b/Cnot8 Caf1z/Toe1 Pan2 Pde12
Rel
ativ
e ex
pres
sion
PapPap Pabpn1 Pabpc1 Pabpc4
0
0.5
1
0 12 24 12 24
0
0.5
1
ZT 0 12 24 12 24 0 12 24 12 24
0
2
4
0
2
4
0 12 24 12 24
0
0.5
1
0 12 24 12 24
ZT
Kojima_Supplemental Fig. 8
Supplemental Fig. 8. The steady-state mRNA expression levels of mouse polymerases and
deadenylases, measured by qPCR and normalized by 36B4. All the graphs (means ± SEM, n=3 for
each time point) are double-plotted for easier visualization of rhythms. Graphs with black circles
and solid lines represent deadenylases with statistically significant mRNA rhythmicity, while graphs
with gray circles and dotted lines show non-rhythmic mRNAs. Rhythmicity was calculated by
Circwave v1.4.
Supplemental Table 3. Oligo DNAs and restriction enzymes used for LM-PAT assay
Gene Sequence Restriction enzyme
Cyp2c55 5’- GGAGTTATTAACTGAAGAGTCATTTTTCAGG -3’ EcoRV
Eif4ebp3 5’- CCACCCTCCAAGTTGGAGTTGC -3’ SacI
Gstm4 5’- AGAGGCTGAGTTCACAGGGA -3’ PstI
Fkbp4 5’- GTGTCTCCGTGCAGGTGCTA -3’ XbaI
Cyp2a4 5’- GTTGATCCTGGGCTGCATGAGG -3’ AccI
Hsd17b6 5’- CACTAGCTATCTGCGCAGACACGC -3’ BsaI
Polr2h 5’- GAAGAAGCTGGCCTTCTGAACTTCG -3’ AciI
Hspb1 5’- AGTCACGCAGTCAGCGGAGAT -3’ ApaI
Gstt2 5’- TGGTCCTGGTTCTGCAACAG -3’ NheI
Mif 5’- CAGAACCGCAACTACAGTAAGCTGC -3’ AccI
Slc44a3 5’- CGTGAACTAGAGCACGGAAG -3’ DraI
Pdzk1ip1 5’- CCAGGCTACAGGGAACATAGAGTTG -3’ HaeIII
Asl 5’- TGGTCCGCAAAGGGATGCCA -3’ PstI
Aqp8 5’- TGCTCTGTCCTGAGCATGCT -3’ ApaI
Rdh9 5’- GAGCCTTCAGAGATGGGAAGGT -3’ SpeI
C2 5’- CACCTGGATGGTGTCCTGGACTTTC -3’ SmaI
Abhd14a 5'- CCACCTCCACCAGGAATTATGCAC -3' XhoI
Gstm3 5'- GTGCCAGCCCTCCCTAGAGATA -3' XhoI
Slc22a1 5'- GCGTGGCTCTGCCTGAGACTATT -3' AvaII
Rbm3 5’- CTGAATGTACCTATGAACAATCCGAGTCAAGATC -3’ DdeI
