Supplementary Material for
Genes adapt non-optimal codon usage to create cell-cycle-dependent
oscillations in protein levels
Milana Frenkel-Morgensterna,b,1,2, Tamar Danona, Thomas Christianc, Takao
Igarashic, Lydia Cohena, Ya-Ming Houc, Lars Juhl Jensend
a
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot,
76100, Israel.
b
Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100,
Israel.
c
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson
University, Philadelphia, PA 19107, USA.
d
Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences,
University of Copenhagen, Copenhagen, DK-2200, Denmark.
1
to whom correspondence should be addressed: [email protected] or
[email protected]
2
current address: Structural Biology and BioComputing Programme, Spanish National
Cancer Research Centre (CNIO), Madrid, 28029, Spain.
This document includes:
Supplementary Tables
Supplementary Figures
Supplementary Results
Supplementary Methods
References
1
Table 1: The codon preferences for sets of the cell-cycle-regulated genes of Schizosaccharomyces pombe: 40 cell-cycleregulated genes in the B1 set, 188 genes in the B2 set, and the 500 most significantly oscillating genes in the top-500 set
were analyzed (Jensen et al, 2006b). The amino acids: Ala, Arg, Ile, Leu, Pro, Thr, Ser, Val, which use wobble inosine
tRNA modification, utilize the non-optimal codons with significant preferences in at least two sets of the cell cycle
regulated genes (P-values were calculated using the bootstrap sampling with the same CAI distribution as that of cell cycle
regulated genes).
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
B1
-0.02
0.01
0
0.01
-0.04
0.02
0
0.01
0
0.01
0.01
-0.01
0
0
0.05
-0.05
0.01
-0.01
-0.01
0.01
-0.02
0
0.01
0.01
0.01
-0.01
0.01
0.02
-0.03
-0.01
0
-0.02
0.01
0.02
0
0.02
-0.02
0
0.01
-0.01
0.01
0.01
-0.01
-0.01
0
-0.01
-0.01
0
0.01
0.01
0.02
-0.01
0
-0.01
0
0.03
-0.03
-0.01
-0.02
0.04
-0.01
Preferences
B2
-0.01
0.02
0
-0.01
-0.01
0.03
-0.02
0.01
0.01
-0.02
0.02
-0.02
0
0
0
0
0
0
0.03
-0.03
-0.03
-0.01
0.03
0.01
0.04
-0.04
0.03
-0.01
-0.02
0.01
-0.01
-0.02
0.01
0.01
0
0.02
-0.02
0
0.02
-0.02
-0.01
0.02
-0.02
0.01
0
0.02
-0.02
-0.01
0
0.01
-0.01
0.03
-0.01
-0.01
0
-0.01
0.01
0.02
-0.01
-0.01
0
Top-500
-0.02
0.01
-0.01
0.02
-0.02
0.03
-0.01
0.02
-0.01
-0.01
0.04
-0.04
0.01
-0.01
0
0
-0.01
0.01
0.02
-0.02
-0.03
-0.02
0.04
0.01
0
0
0.01
0.02
-0.03
0.01
-0.02
-0.01
0.01
0.01
0
0.01
-0.01
0
0.04
-0.04
0.01
0.01
-0.01
-0.01
0
0.01
-0.02
0.01
0
0
0.01
0.01
-0.01
-0.01
0
-0.02
0.02
0.01
-0.01
0
0
2
B1
0.14
0.26
0.53
0.29
0.02
0.24
0.51
0.22
0.51
0.27
0.3
0.3
0.51
0.51
0.05
0.05
0.29
0.29
0.5
0.52
0.19
0.52
0.34
0.24
0.34
0.34
0.28
0.13
0.08
0.22
0.52
0.01
0.09
0.06
0.52
0.19
0.19
1
0.32
0.32
0.27
0.27
0.2
0.53
0.53
0.17
0.21
0.45
0.14
0.25
0.01
0.26
0.51
0.19
1
0.07
0.07
0.26
0.1
0.02
0.23
P-values
B2
0.13
0.0001
0.54
0.14
0.14
0.01
0.0001
0.06
0.01
0.0001
0.04
0.04
0.53
0.52
0.51
0.52
0.52
0.53
0.0001
0.0001
0.0001
0.05
0.01
0.07
0.0001
0.0001
0.0001
0.14
0.02
0.05
0.07
0.01
0.0001
0.05
0.53
0.04
0.04
1
0.06
0.06
0.19
0.01
0.05
0.18
0.57
0.0001
0.05
0.05
0.55
0.01
0.19
0.0001
0.18
0.08
1
0.17
0.17
0.0001
0.01
0.14
0.52
Top-500
0.0001
0.02
0.0001
0.0001
0.0001
0.0001
0.0001
0.06
0.0001
0.02
0.0001
0.0001
0.03
0.03
0.52
0.52
0.05
0.05
0.0001
0.0001
0.0001
0.0001
0.0001
0.01
0.52
0.53
0.02
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.01
0.56
0.08
0.08
1
0.0001
0.0001
0.02
0.02
0.0001
0.55
0.59
0.0001
0.0001
0.1
0.58
0.58
0.01
0.04
0.09
0.03
1
0.0001
0.0001
0.02
0.02
0.53
0.54
Table 2: The codon preferences for sets of the cell-cycle-regulated genes of Saccharomyces cerevisiae: 113 cell cycle
regulated genes in the B1 set, 352 genes in the B2 set, and the 600 most significantly oscillating genes in the top-600 set
were studied (Jensen et al, 2006b). The amino acids: Ala, Arg, Ile, Pro, Thr, Ser, Val, which use wobble inosine tRNA
modification, utilize the non-optimal codons with significant preferences in at least two sets of the cell cycle regulated genes
(P-values were calculated using the bootstrap sampling with the same CAI distribution as that of cell cycle regulated
genes).
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
B1
-0.02
0
-0.02
0.04
0.02
0.01
-0.01
0.01
-0.02
-0.01
-0.01
0.01
-0.03
0.03
0.04
-0.04
-0.02
0.02
-0.03
0.03
-0.03
-0.01
0.06
-0.02
-0.04
0.04
-0.01
0.02
-0.01
-0.01
0.02
0.02
-0.01
-0.01
-0.01
-0.01
0.01
0
0.02
-0.02
0.04
0.01
-0.01
-0.04
-0.01
0
0
-0.01
-0.02
0.03
0.03
-0.02
0.01
-0.02
0
0
0
0
0
0.01
-0.01
Preferences
B2
-0.01
0.01
0
0
-0.01
-0.01
0.01
0.01
0
0
0.02
-0.02
0.02
-0.02
-0.03
0.03
0.01
-0.01
0.01
-0.01
-0.01
-0.01
0.01
0.01
-0.01
0.01
0.01
-0.01
0
0
-0.01
0
0
0
0.01
0
0
0
0.01
-0.01
0.01
-0.01
0.01
-0.02
0
0.01
-0.01
-0.01
0.01
0
0
0.02
-0.02
0
0
-0.02
0.02
0.01
-0.01
-0.01
0.01
Top-600
-0.03
0.01
0
0.02
-0.01
0
-0.01
0.01
0.01
0
0.03
-0.03
0.02
-0.02
0
0
0.02
-0.02
0
0
-0.03
0
0.03
0
-0.01
0.01
0.01
0.02
-0.03
-0.01
-0.02
0
0
0.02
0.01
0
0
0
0.03
-0.03
0.02
0.01
-0.01
-0.02
0
0.01
-0.01
-0.01
0.01
0.01
0.03
0.02
-0.04
-0.01
0
-0.04
0.04
0.02
-0.02
-0.01
0.01
3
B1
0.06
0.52
0.12
0.0001
0.09
0.001
0.52
0.52
0.001
0.09
0.21
0.21
0.001
0.001
0.04
0.04
0.07
0.07
0.001
0.001
0.01
0.12
0.01
0.02
0.01
0.01
0.18
0.05
0.25
0.06
0.01
0.04
0.01
0.18
0.05
0.22
0.22
1
0.08
0.08
0.01
0.52
0.13
0.0001
0.03
0.53
0.52
0.08
0.001
0.001
0.0001
0.02
0.21
0.01
1
0.51
0.52
0.51
0.52
0.22
0.16
P-values
B2
0.09
0.04
0.53
0.52
0.12
0.05
0.06
0.55
0.56
0.54
0.0001
0.0001
0.0001
0.0001
0.01
0.01
0.11
0.11
0.06
0.06
0.1
0.02
0.05
0.05
0.52
0.52
0.06
0.07
0.52
0.55
0.03
0.55
0.58
0.54
0.0001
0.53
0.52
1
0.12
0.12
0.51
0.0001
0.02
0.06
0.57
0.01
0.02
0.01
0.01
0.53
0.53
0.05
0.0001
0.54
1
0.01
0.01
0.05
0.07
0.09
0.05
Top-600
0.0001
0.02
0.55
0.0001
0.07
0.54
0.54
0.57
0.0001
0.55
0.0001
0.0001
0.0001
0.0001
0.52
0.52
0.0001
0.0001
0.54
0.55
0.0001
0.55
0.02
0.54
0.09
0.09
0.03
0.0001
0.0001
0.0001
0.0001
0.57
0.59
0.0001
0.0001
0.53
0.53
1
0.0001
0.0001
0.0001
0.01
0.55
0.0001
0.58
0.0001
0.01
0.0001
0.0001
0.01
0.0001
0.0001
0.0001
0.01
1
0.0001
0.0001
0.0001
0.0001
0.05
0.02
Table 3: The codon preferences for sets of the cell-cycle-regulated genes of Arabidopsis thaliana: 61 cell cycle
regulated genes in the B1 set, 176 genes in the B2 set (Jensen et al, 2006b). The top-400 set was excluded from the
analysis because of the poor quality of the DNA microarray expression data, which was inconsistent with other sets.
Codons characterized by the positive consistent bias in two sets are presented in bold. These codons correspond to
non-optimal codons of amino acids encoded by two codons (His, Cys, Phe, Asn, Asp, Tyr) and for those using
wobble inosine tRNA modifications (namely, Ala, Arg, Ile, Pro, Ser, Thr, Val: P-values were calculated using the
bootstrap sampling with the same CAI distribution as that of cell cycle regulated genes).
Preferences
P-values
Aa
Codon
5'->3'
B1
B2
B1
B2
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
0.01
-0.03
-0.01
0.03
0.05
-0.01
-0.01
-0.01
-0.01
-0.01
-0.01
0.01
-0.02
0.02
0.06
-0.06
0.01
-0.01
0.01
-0.01
0.05
-0.01
0
-0.04
-0.02
0.02
-0.02
0.04
-0.02
0.01
0.01
0
-0.01
-0.02
0.01
0.03
-0.03
0
-0.02
0.02
0.05
-0.02
-0.02
-0.01
0.01
-0.03
-0.01
-0.01
0
0.04
0.01
0.03
-0.02
-0.03
0.02
0.01
-0.01
0.01
0
-0.01
0
-0.06
0.06
-0.03
0.03
0
0
0.04
-0.04
-0.03
0.03
-0.01
0.01
-0.01
0.01
-0.03
0.03
-0.05
0.03
0.02
0
0
0
-0.03
0.01
0.02
0.01
-0.01
0
-0.05
0.05
0.01
0.01
-0.03
0.01
-0.02
-0.01
0.02
0.01
-0.01
0.01
0.04
0.25
0.01
0.22
0.03
0.01
0.22
0.24
0.11
0.14
0.16
0.01
0.01
0.02
0.02
0.01
0.01
0.33
0.33
0.28
0.28
0.0001
0.2
0.5
0.0001
0.01
0.01
0.14
0.01
0.1
0.18
0.13
0.52
0.21
0.03
0.13
0.04
0.04
1
0.01
0.01
0.02
0.03
0.12
0.29
0.14
0.0001
0.18
0.17
0.52
0.0001
0.27
0.0001
0.0001
0.0001
0.15
0.52
0.09
0.11
0.54
0.03
0.53
0.0001
0.0001
0.0001
0.0001
0.51
0.51
0.0001
0.0001
0.0001
0.0001
0.13
0.08
0.13
0.53
0.02
0.0001
0.0001
0.0001
0.13
0.53
0.54
0.54
0.0001
0.06
0.1
0.16
0.16
1
0.0001
0.0001
0.01
0.01
0.0001
0.19
0.0001
0.03
0.0001
0.05
0.03
0.01
0.0001
4
Aa
Codon
5'->3'
Preferences
P-values
Aa
Codon
5'->3'
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
-0.04
0.04
-0.01
0
-0.06
0.06
-0.04
0
0.04
0
-0.03
0.02
-0.03
0
0.04
-0.04
-0.01
0.01
0.01
-0.01
0.25
0.01
0.25
1
0.02
0.02
0.1
0.51
0.0001
0.51
0.52
0.02
0.0001
1
0.0001
0.0001
0.09
0.06
0.01
0.1
5
Table 4: The codon preferences for sets of cell cycle regulated genes in Homo sapiens: 63 cell cycle regulated genes in
the B1 set, 438 genes in the B2 set, and the 600 most significantly oscillating genes in the top-600 set were analyzed
(Jensen et al, 2006b). The GC content distribution was preserved in the bootstrap sampling to calculate the p-values.
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
B1
0.04
-0.1
-0.01
0.07
0.07
-0.02
0
-0.01
-0.06
0.02
-0.13
0.13
-0.1
0.1
-0.15
0.15
0.1
-0.1
0.13
-0.13
0.03
-0.04
-0.05
0.05
-0.14
0.14
0.05
-0.12
0.06
0.02
-0.05
-0.1
0.03
0.06
0.04
0.04
-0.04
0
-0.13
0.13
0.07
-0.1
-0.02
0.05
-0.05
0.03
0.03
-0.05
-0.02
0.07
0.14
-0.04
-0.1
0.02
-0.05
-0.05
0.09
0
-0.08
0.08
0.07
Preferences
B2
0.04
-0.07
-0.03
0.05
0.06
-0.02
0.03
-0.04
-0.04
0.02
-0.11
0.11
-0.1
0.1
-0.12
0.12
0.06
-0.06
0.1
-0.1
0.05
-0.06
-0.03
0.04
-0.13
0.13
0.05
-0.12
0.08
0.02
-0.04
-0.08
0.04
0.04
0.03
0.09
-0.09
0
-0.1
0.1
0.04
-0.06
-0.03
0.05
-0.05
0.04
0.03
-0.04
-0.01
0.04
0.03
-0.04
0
0.04
-0.07
-0.03
0.07
0
-0.1
0.1
0.05
Top-600
0.03
-0.04
-0.02
0.03
0.05
-0.01
0.02
-0.03
-0.03
0.01
-0.08
0.08
-0.07
0.07
-0.04
0.04
0.05
-0.05
0.08
-0.08
0.03
-0.04
-0.03
0.03
-0.07
0.07
0.04
-0.08
0.04
0.01
-0.03
-0.06
0.03
0.03
0.02
0.06
-0.06
0
-0.07
0.07
0.04
-0.06
-0.02
0.04
-0.03
0.03
0.02
-0.03
-0.01
0.03
0.04
0.0001
-0.04
0.02
-0.05
-0.02
0.04
0
-0.06
0.06
0.04
6
B1
0.0527
0.0001
0.809
0.0004
0.015
0.0264
0.727
0.8357
0.0011
0.016
0.0001
0.0001
0.0016
0.0006
0.0001
0.0001
0.0002
0.0001
0.0011
0.0015
0.3096
0.1505
0.0043
0.0096
0.0001
0.0001
0.0045
0.0002
0.0092
0.0092
0.0001
0.0001
0.0338
0.0001
0.0001
0.2291
0.2455
1
0.0001
0.0001
0.0048
0.0003
0.4117
0.0132
0.002
0.0218
0.0356
0.0004
0.0052
0.0001
0.0117
0.2354
0.0613
0.3576
0.0391
0.001
0.0001
1
0.0102
0.0101
0.0001
P-values
B2
0.1704
0.0007
0.0104
0.0022
0.4612
0.0001
0.0001
0.4426
0.2556
0.0001
0.0001
0.0001
0.0002
0.0002
0.0001
0.0001
0.3992
0.4106
0.0257
0.0244
0.1902
0.0048
0.097
0.0013
0.0001
0.0001
0.0048
0.0001
0.0001
0.0007
0.0001
0.0092
0.0003
0.0063
0.0001
0.0013
0.0011
1
0.0001
0.0001
0.1377
0.0098
0.0336
0.0007
0.0003
0.0001
0.0615
0.0005
0.6396
0.0001
0.7439
0.0832
0.0908
0.1263
0.0004
0.0284
0.0001
1
0.0001
0.0001
0.0001
Top-600
0.075
0.1164
0.0194
0.0471
0.0666
0.0106
0.0001
0.1833
0.1305
0.0101
0.0001
0.0001
0.0023
0.0022
0.5953
0.6024
0.082
0.0835
0.0064
0.0048
0.4594
0.0267
0.0039
0.001
0.0104
0.0096
0.0012
0.0023
0.0812
0.2095
0.0001
0.0071
0.0011
0.0039
0.0057
0.0291
0.0275
1
0.0008
0.0007
0.0051
0.0001
0.0498
0.0002
0.0489
0.0001
0.1758
0.0009
0.0956
0.0005
0.3215
0.3323
0.2014
0.7048
0.0048
0.0856
0.004
1
0.0221
0.0244
0.0001
Table 5: The codon preferences for sets of cell cycle regulated genes in Schizosaccharomyces pombe: 40 cell cycle regulated
genes in the B1 set, 188 genes in the B2 set, and the 500 most significantly oscillating genes in the top-500 set were analyzed
(Jensen et al, 2006b). The GC content distribution was preserved in the bootstrap sampling to calculate the p-values.
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
B1
-0.02
0.01
0
0.01
-0.04
0
0.01
0.01
0
0.02
0.01
-0.01
0
0
-0.05
0.05
-0.01
0.01
0.01
-0.01
-0.02
0.01
0
0.01
0.01
-0.01
-0.03
0.01
0.02
-0.02
0.01
0
-0.01
0
0.02
-0.02
0.02
0
0.01
-0.01
0.01
0.01
-0.01
-0.01
0.01
0
-0.01
-0.01
0
0.01
0
-0.01
-0.01
0.02
0
-0.03
0.03
-0.02
-0.01
-0.01
0.04
Preferences
B2
-0.01
0.02
0
-0.01
-0.01
-0.02
-0.02
0.01
0.01
0.03
0.02
-0.02
0
0
0
0
0
0
-0.03
0.03
-0.03
0.01
-0.01
0.03
0.04
-0.04
-0.02
0.03
-0.01
-0.02
0.01
0
0.01
-0.01
0.01
-0.02
0.02
0
0.02
-0.02
-0.01
0.02
-0.02
0.01
0
-0.01
-0.02
0.02
0
0.01
-0.01
0.03
-0.01
-0.01
0
0.01
-0.01
-0.01
0.02
0
-0.01
Top-500
-0.02
0.01
-0.01
0.02
-0.02
-0.01
-0.01
0.02
-0.01
0.03
0.04
-0.04
0.01
-0.01
0
0
0.01
-0.01
-0.02
0.02
-0.03
0.01
-0.02
0.04
0
0
-0.03
0.01
0.02
-0.01
0.01
0
0.01
-0.02
0.01
-0.01
0.01
0
0.04
-0.04
0.01
0.01
-0.01
-0.01
0
0.01
-0.02
0.01
0
0
-0.01
0.01
-0.01
0.01
0
0.02
-0.02
-0.01
0.01
0
0
7
B1
0.6316
0.7494
0.179
0.6073
0.2313
0.0632
0.0242
0.6517
0.2436
0.9277
0.7878
0.7833
0.7679
0.2583
0.0125
0.0096
0.2501
0.2493
0.0147
0.0144
0.6574
0.3953
0.0733
0.8599
0.5895
0.5897
0.757
0.8638
0.4069
0.0811
0.3533
0.382
0.021
0.0134
0.421
0.9296
0.9272
1
0.8409
0.8339
0.022
0.7195
0.4835
0.0868
0.2397
0.3179
0.7458
0.0133
0.3912
0.4856
0.0503
0.0249
0.3604
0.3611
1
0.0085
0.0063
0.455
0.0182
0.4457
0.0335
P-values
B2
0.7231
0.1097
0.1672
0.0217
0.5691
0.0076
0.0659
0.2516
0.0006
0.3318
0.2377
0.2475
0.8491
0.1858
0.7252
0.2946
0.694
0.3507
0.0131
0.012
0.0794
0.1447
0.3866
0.2426
0.0134
0.0178
0.234
0.0054
0.0803
0.0001
0.0196
0.3658
0.1753
0.4817
0.2968
0.6081
0.6158
1
0.2681
0.2579
0.6735
0.0796
0.0038
0.437
0.6051
0.132
0.0092
0.0019
0.4407
0.2375
0.6753
0.0125
0.1964
0.0778
1
0.5592
0.5625
0.5166
0.1312
0.2265
0.0686
Top-500
0.372
0.7791
0.0904
0.0642
0.4486
0.7824
0.76
0.0072
0.0224
0.8349
0.0007
0.0005
0.3678
0.3651
0.7853
0.2442
0.0847
0.0879
0.2173
0.2075
0.065
0.1933
0.0074
0.041
0.9573
0.051
0.2906
0.9313
0.0518
0.0748
0.0146
0.2546
0.3646
0.1286
0.5439
0.9999
0.9997
1
0.0023
0.0017
0.0006
0.7913
0.2516
0.0025
0.8208
0.0479
0.006
0.198
0.3446
0.9103
0.9256
0.8532
0.1278
0.4456
1
0.0968
0.1008
0.6921
0.7683
0.0985
0.8081
Table 6: The codon preferences for sets of cell cycle regulated genes in Saccharomyces cerevisiae: 113 cell cycle
regulated genes in the B1 set, 352 genes in the B2 set, and the 600 most significantly oscillating genes in the top-600
set were studied (Jensen et al, 2006b). The GC content distribution was preserved in the bootstrap sampling to calculate
the p-values calculation.
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
B1
-0.02
0
-0.02
0.04
0.02
-0.01
-0.01
0.01
-0.02
0.01
-0.01
0.01
-0.03
0.03
-0.04
0.04
0.02
-0.02
0.03
-0.03
-0.03
-0.02
-0.01
0.06
-0.04
0.04
-0.01
-0.01
0.02
0.02
-0.01
-0.01
-0.01
0.02
-0.01
0.01
-0.01
0
0.02
-0.02
0.04
0.01
-0.01
-0.04
-0.02
-0.01
0
0
-0.01
0.03
0.01
-0.02
-0.02
0.03
0
0
0
0
0
-0.01
0.01
Preferences
B2
-0.01
0.01
0
0
-0.01
0.01
0
0.01
0
-0.01
0.02
-0.02
0.02
-0.02
0.03
-0.03
-0.01
0.01
-0.01
0.01
-0.01
0.01
-0.01
0.01
-0.01
0.01
0
0.01
-0.01
0
0
0.01
0
-0.01
0
0
0
0
0.01
-0.01
0.01
-0.01
0.01
-0.02
0.01
-0.01
-0.01
0.01
0
0
-0.02
0.02
0
0
0
0.02
-0.02
-0.01
0.01
0.01
-0.01
Top-600
-0.03
0.01
0
0.02
-0.01
-0.01
0
0.01
0.01
0
0.03
-0.03
0.02
-0.02
0
0
-0.02
0.02
0
0
-0.03
0
0
0.03
-0.01
0.01
-0.03
0.01
0.02
0
0
0.01
-0.01
-0.02
0.02
0
0
0
0.03
-0.03
0.02
0.01
-0.01
-0.02
0.01
-0.01
-0.01
0.01
0
0.01
-0.04
0.02
-0.01
0.03
0
0.04
-0.04
-0.02
0.02
0.01
-0.01
8
B1
0.0537
0.4895
0.0039
0.0038
0.0663
0.1901
0.0681
0.0255
0
0.1914
0.1307
0.1326
0.0008
0.0007
0.0317
0.0306
0.0511
0.0483
0.001
0.0005
0.0058
0.016
0.1177
0.0022
0.0034
0.0031
0.3001
0.0801
0.04
0.0001
0.0059
0.0304
0.0421
0.0032
0.1332
0.1268
0.1208
1
0.0628
0.0584
0.0052
0.1238
0.1133
0.0002
0.0002
0.0912
0.4554
0.6053
0.0251
0.0021
0.1557
0.006
0.0117
0.0062
1
0.5929
0.4449
0.4714
0.5678
0.1653
0.224
P-values
B2
0.6911
0.4651
0.394
0.7583
0.1495
0.0203
0.2864
0.022
0.6645
0.0152
0.4143
0.4046
0.265
0.2658
0.0167
0.0176
0.3049
0.3042
0.2508
0.2561
0.6334
0.1417
0.0346
0.5185
0.005
0.0039
0.0478
0.8303
0.1334
0.4506
0.7636
0.0862
0.3341
0.6879
0.9336
0.0038
0.998
1
0.7429
0.7441
0.2361
0.0136
0.0186
0.0084
0.0207
0.103
0.4314
0.2443
0.71
0.7545
0.1129
0.0667
0.5276
0.5613
1
0.5611
0.5524
0.538
0.5708
0.1643
0.2226
Top-600
0.0001
0.2998
0.5848
0.0062
0.1889
0.1001
0.3591
0.0095
0.0004
0.7963
0.0045
0.0034
0.1109
0.1081
0.6374
0.3994
0.0309
0.0306
0.1333
0.9044
0.0009
0.8372
0.5272
0.0093
0.001
0.0011
0.0016
0.803
0
0.4183
0.8485
0.0575
0.0026
0.0167
0.0078
0.0022
0.9993
1
0.0045
0.0037
0.005
0.0624
0.0056
0.0007
0.0064
0.0356
0.2991
0.1216
0.7872
0.0458
0
0.0152
0.0128
0
1
0.0006
0.0007
0.0082
0.0047
0.1353
0.2169
Table 7: The codon preferences for sets of cell cycle regulated genes in Arabidopsis thaliana: 61
cell cycle regulated genes in the B1 set, 176 genes in the B2 set (Jensen et al, 2006b). The GC
content distribution was preserved in the bootstrap sampling to calculate the p-values.
Aa
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Codon
5'->3'
GCA
GCC
GCG
GCT
AGA
CGT
AGG
CGC
CGG
CGA
AAC
AAT
GAC
GAT
TGT
TGC
CAG
CAA
GAG
GAA
GGA
GGG
GGT
GGC
CAC
CAT
ATC
ATT
ATA
CTT
TTA
CTA
CTC
TTG
CTG
AAG
AAA
ATG
TTC
TTT
CCA
CCC
CCG
CCT
TCG
TCC
TCA
AGT
AGC
TCT
ACT
Preferences
B1
B2
0.01
-0.03
-0.01
0.03
0.05
-0.01
-0.01
-0.01
-0.01
-0.01
-0.01
0.01
-0.02
0.02
-0.06
0.06
-0.01
0.01
-0.01
0.01
0.05
-0.04
-0.01
0
-0.02
0.02
-0.02
-0.02
0.04
0
-0.01
0.01
0.01
0.01
-0.02
-0.03
0.03
0
-0.02
0.02
0.05
-0.02
-0.02
-0.01
0
-0.01
-0.01
-0.03
0.01
0.04
0.04
0.03
-0.02
-0.03
0.02
0.01
0.01
0
0
-0.01
-0.01
-0.06
0.06
-0.03
0.03
0
0
-0.04
0.04
0.03
-0.03
-0.01
0.01
0.01
-0.01
-0.03
0.03
0.02
-0.05
0.03
0
-0.03
0.02
0
0
0.01
-0.01
0.01
0
-0.05
0.05
0.01
0.01
-0.03
0.01
-0.01
0.01
0.02
-0.01
-0.02
0.01
0.02
9
P-values
B1
0.5811
0.027
0.4532
0.0497
0.0095
0.1914
0.1339
0.2345
0.252
0.4042
0.6493
0.6421
0.3741
0.3775
0.012
0.012
0.1606
0.162
0.0631
0.0619
0.0003
0.0001
0.1053
0.5212
0.4925
0.5024
0.0117
0.3698
0.009
0.613
0.5832
0.0906
0.1899
0.2466
0.0041
0.0073
0.0067
1
0.4079
0.4177
0.0088
0.0313
0.3042
0.1197
0.4054
0.0299
0.0609
0.0014
0.0366
0.0003
0.0287
B2
0.0038
0.0266
0.0001
0.0411
0.4378
0.126
0.5163
0.2848
0.0495
0.2432
0.0001
0.0001
0.0081
0.0076
0.3774
0.6434
0.0002
0.0001
0.0023
0.0025
0.0916
0.0076
0.1375
0.1134
0.0843
0.0833
0.0312
0.0001
0.0024
0.6046
0.0001
0.0003
0.6876
0.6687
0.0903
0.0875
0.0892
1
0.0003
0.0002
0.2659
0.0247
0.0094
0.4257
0.028
0.1365
0.0034
0.1903
0.0003
0.1132
0.1158
Aa
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
Codon
5'->3'
ACC
ACA
ACG
TGG
TAT
TAC
GTC
GTA
GTT
GTG
Preferences
P-values
-0.04
-0.01
0.01
0
0.06
-0.06
0
-0.04
0
0.04
10
-0.03
-0.03
0.04
0
-0.04
0.04
0.01
-0.01
-0.01
0.01
Aa
0.0039
0.5099
0.4218
1
0.0005
0.0002
0.7828
0.0011
0.3464
0.0055
Codon
5'->3'
0.0004
0.0003
0.0001
1
0.0162
0.0161
0.1722
0.3
0.1812
0.2979
Table 8: The codon preferences for the cell cycle regulated genes in Homo Sapiens and Saccharomyces
cerevisiae expressed in G1 phase of the cell cycle, the top-600 genes are used for the analysis (Jensen et al,
2006b). The first 40 codons as well as the full sequences of these genes adopt mostly optimal codons.
Aa
Codon
5'->3'
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
GCA
GCC
GCG
GCT
AGA
AGG
CGA
CGC
CGG
CGT
AAC
AAT
GAC
GAT
TGC
TGT
CAA
CAG
GAA
GAG
GGA
GGC
GGG
GGT
CAC
CAT
ATA
ATC
ATT
CTA
CTC
CTG
CTT
TTA
TTG
AAA
AAG
ATG
TTC
TTT
CCA
CCC
CCG
CCT
AGC
AGT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
TGG
TAC
TAT
GTA
GTC
GTG
GTT
Human
G1 phase
40 first
genes
codons
-0.002
-0.088
-0.004
0.014
-0.012
0.146
0.017
-0.073
0
-0.082
0.002
-0.022
0.01
-0.013
0
0.12
-0.014
0.017
0.002
-0.021
-0.009
0.123
0.009
-0.123
-0.004
0.078
0.004
-0.078
0.001
0.205
-0.001
-0.205
-0.021
-0.054
0.021
0.054
0.019
-0.131
-0.019
0.131
-0.014
-0.082
0.011
0.128
-0.019
-0.006
0.021
-0.041
0.009
0.094
-0.009
-0.094
0.019
-0.002
-0.023
0.057
0.004
-0.055
0.002
-0.001
-0.011
0.05
-0.006
-0.037
-0.001
-0.019
0.008
-0.019
0.007
0.026
0.002
-0.102
-0.002
0.102
0
0
-0.036
0.166
0.036
-0.166
-0.001
-0.095
-0.014
0.003
0.003
0.205
0.011
-0.113
-0.009
0.016
0.001
-0.051
-0.009
-0.078
0.001
0.044
0.007
0.098
0.008
-0.029
-0.005
-0.128
0.008
0.082
-0.006
0.08
0.004
-0.034
0
0
-0.011
0.09
0.011
-0.09
0.018
-0.017
-0.015
0.051
-0.007
0.037
0.002
-0.072
11
Yeast
G1 phase
40 first
genes
codons
-0.015
-0.028
0.005
0.022
-0.001
0.021
0.012
-0.014
-0.002
0.023
-0.008
-0.046
-0.02
0.007
0.01
0.037
-0.009
-0.012
0.031
-0.007
0.013
0.045
-0.013
-0.045
0.003
0.003
-0.003
-0.003
-0.023
0.009
0.023
-0.009
0.005
-0.071
-0.005
0.071
0.003
-0.034
-0.003
0.034
-0.048
-0.051
0.005
0.04
-0.007
0.008
0.05
0.003
-0.006
0.022
0.006
-0.022
-0.049
-0.032
0.023
0.014
0.026
0.017
0.009
0.032
-0.003
0.06
-0.005
-0.016
-0.006
0.022
-0.015
-0.038
0.021
-0.059
-0.014
-0.03
0.014
0.03
0
0
0.054
0.082
-0.054
-0.082
0.024
0.029
0.009
0.044
-0.008
0.006
-0.026
-0.079
-0.006
0.016
-0.011
-0.02
-0.007
0.016
0.014
0.014
-0.009
0.006
0.019
-0.032
-0.024
-0.083
0.004
0.016
-0.011
0.029
0.03
0.038
0
0
0.024
0.028
-0.024
-0.028
-0.03
0.001
0.014
0.013
0.003
0.014
0.013
-0.027
Table 9: The codon-bias scores for two sets of proteins: cell cycle and non-cell
cycle regulated according to protein dynamics: 17 genes are from a previous study
(Sigal et al, 2006). The genes depicted in blue were produced for the current study.
Gene Name
Classification*
CCCS score**
DDX5
CCR
0.022
USP7
CCR
0.011
TOP1
CCR
0.002
ANP32B
CCR
0.011
H2AFV
CCR
0.008
GTF2F2
CCR
0.02
RBBP7
CCR
0.014
SFRS10
CCR
0.012
GARS
CCR
0.007
TARS
CCR
0.012
EPRS
CCR
0.021
SAE1
NCCR
-0.002
SET
NCCR
0.02
HMGA2
NCCR
-0.012
YPEL1
NCCR
-0.024
DDX46
NCCR
0.018
LMNA
NCCR
-0.03
HMGA1
NCCR
-0.018
ZNF433
NCCR
-0.013
KIAA1937
NCCR
-0.012
WARS
NCCR
-0.015
GAPDH
NCCR
-0.026
* CCR represents the protein with cell-cycle-regulated protein dynamics; NCCR is non-cell-cycleregulated genes. ** CCCS is the Cell-Cycle Codon score.
12
Table 10: Comparison of the non-optimal codon abundance for human cell cycle
regulated genes from B1 set with that for the non-cell cycle regulated paralogs. The
criterion for finding the appropriate cases was to use only well-known cell cycle
genes with clearly cycling mRNA for which obvious paralogs with clearly noncycling mRNA levels exist.
Cell Cycle
Percentage of Non-
Non-Cell Cycle
Percentage of Non-
Regulated Gene
Optimal Codons
Regulated Paralogs
Optimal Codons
STAG1
60%
STAG3
42%
ANP32E
60%
ANP32B
46%
EZH2
57%
KIAA0388
33%
E2F5
53%
E2F4
31%
13
Supplementary Figures
Figure 1: Cell cycle dynamics of six proteins during two generations. A. Glutamylprolyl-tRNA synthetase (EPRS). B. Threonyl-tRNA synthetase (TARS). C. Glycyl-tRNA
synthetase (GARS). D. Arginine and glutamate-rich protein 1. E. Tryptophanyl-tRNA
synthetase (WARS). F. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A-D.
Proteins with cell cycle dependent protein dynamics. E-F. Non-cell cycle dependent
proteins.
14
15
Figure 2: The dynamics of ARGLU1 mRNA expression is very similar to its protein dynamics
found using the time-lapse microscopy of the YFP-tagged clone. A. ARGLU1 mRNA
expression as seen in two different experiments in Whitfield et al (Whitfield et al, 2002). ARGLU1
expression rises during the G2 phase and likewise, B. ARGLU1 protein dynamics peaks in G2.
16
Supplementary Results
To test if the observed codon preferences were associated with a particular cell cycle
phase, we partitioned the top-600 cycling genes according to their time of peak
expression and searched for codon preferences that distinguished cycling genes with
different peak times. The strongest codon preference was observed when comparing
genes expressed during S, G2 and M phase to genes expressed during G1 phase (less than
13-20% of genes from the top-600 group). It should be noted that the late G1 phase
corresponds to 20% to 40% of the cell cycle in yeast (123 genes), whereas in humans the
G1 phase ranges from 5% to 40% of the cell cycle (78 genes). Notably, this comparison
yielded almost exactly the same codon preferences as when cell cycle regulated and noncell cycle regulated genes were compared.
To ensure that the observed bias is linked to cell cycle regulation and not cell cycle
function in general, we analyzed the codon preference of non-cycling genes with a cell
cycle phenotype at the mRNA level (Mukherji et al, 2006) and that of non-cycling genes
with cell cycle regulated orthologs (Jensen et al, 2006a). Neither of these gene sets
identified the preference for non-optimal codon usage observed for cell cycle regulated
genes. That fact strongly suggests that the observed codon preference is specifically
related to cyclic expression and not to cell growth or cell proliferation in general.
The charged tRNA levels presumably peak in G2/M or M phase. Thus, codon preference
maximally affects a phase of the cell cycle when a very large number of proteins are
produced. Codon preference may not serve to ensure that genes are expressed at a
specific time point but rather, it may primarily be a mechanism to ensure that they are not
expressed inappropriately. In other words, to ensure that genes needed for cell cycle
progression are not accidentally expressed in starving cells resting in G1 (or G0) phase.
17
Supplementary Methods
1. Construction of the tagged protein library
A library of fluorescently tagged proteins was constructed in a non-small cell lung
carcinoma cell line (H1299) by a two stage process. In both stages, a fluorescent reporter
was integrated into the genome via Central Dogma tagging (CD-tagging: Jarvik et al,
1996; Sigal et al, 2007). In the first stage a parental clone was produced in which the
nucleus is colored brighter than the cytoplasm and the cytoplasm is colored brighter than
the background image. For this purpose, a red fluorescent protein (mCherry) was
introduced in two rounds of CD-tagging. In the first round, the H7a clone expressing the
tagged XRCC5 protein localized to the nucleus was selected. In the second round (carried
out on the previously selected clone H7a), clone H7 expressing tagged DAP1 localized to
the whole intracellular domain was selected. Following these two steps, a parental clone
was obtained that expresses two mCherry endogenously tagged proteins (XRCC5 and
DAP1), one staining the cytoplasm and the other more intensely staining the nucleus. We
found that the cell-cell variability of red fluorescence in this clone was much less than
that among the clones generated by transfection of mCherry, which was crucial for
reliable image analysis of the cell videos.
The second stage in the generation of the library was to use CD-tagging in order to tag
different proteins in the parental H7 clone (H1299-cherry) with eYFP or Venus (Sigal et
al, 2007: the CD-tagging protocol used is described in detail by (Sigal et al. 2007).
Briefly, a fluorescent protein (FP), flanked by splice acceptor and donor sequences was
integrated into the genome as an artificial exon via retroviral vectors (U5000, U5001,
U5002), each containing FP in one of 3 reading frames. Cells positive for relevant FP
fluorescence were sorted into 384 well plates by flow cytometry and expanded into cell
clones. Tagged protein identities were determined by 3’RACE, using a nested PCR
reaction that amplified the section between the FP and the host gene’s polyA mRNA tail.
The PCR product was sequenced directly and aligned to the genome. Our library of CDtagged proteins is detailed at www.dynamicproteomics.net.
18
2. Long period time-lapse microscopy
Time-lapse movies were obtained (at 20x magnification) as described elsewhere (Sigal et
al. 2007) with an automated, humidity and CO2 controlled Leica DMIRE2 inverted
fluorescence microscope and an ORCA ER cooled CCD camera (Hamamatsu Photonics).
The system was controlled by ImagePro5 Plus (Media Cybernetics) software which
integrated time-lapse acquisition, stage movement and software based auto-focus. During
the experiment, the cells were grown and visualized in 12-well coverslip bottom plates
(MatTek) coated with 10μM fibronectin (Sigma). In each well (15 or more cells), time
lapse movies were obtained from four fields and each movie was taken at a time
resolution of 20 minutes, filmed for at least three days (over 200 time points). Each time
point included three images: phase contrast, red and yellow fluorescence.
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