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Title 長鎖ノンコーディングRNAによる遺伝子発現制御と生物 多様化機構

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Title 長鎖ノンコーディングRNAによる遺伝子発現制御と生物 多様化機構
Title
Author(s)
Citation
Issue Date
長鎖ノンコーディングRNAによる遺伝子発現制御と生物
多様化機構( Dissertation_全文 )
上坂, 将弘
Kyoto University (京都大学)
2015-03-23
URL
https://doi.org/10.14989/doctor.k18832
Right
許諾条件により本文は2016/03/23に公開
Type
Thesis or Dissertation
Textversion
ETD
Kyoto University
RNA
1
緒言 ............................................................................................................. 3
第 1 章 一本鎖 lncRNA、pancRNA による遺伝子発現制御の普遍性 .......................... 11
序論 ......................................................................................................................................... 11
材料と方法 .............................................................................................................................. 14
結果 ......................................................................................................................................... 25
考察 ......................................................................................................................................... 56
第 2 章 pancRNA を介した遺伝子発現制御の種間多様性 ......................................... 62
序論 ......................................................................................................................................... 62
材料と方法 .............................................................................................................................. 66
結果 ......................................................................................................................................... 80
考察 ....................................................................................................................................... 108
総括 ......................................................................................................... 122
謝辞 ......................................................................................................... 128
リファレンス .............................................................................................. 130
2
緒言
DNA
DNA
(Kuratani, 2004)
(Carroll et al., 2009)
DNA
(Kimura, 1968)
1990
2000
(Lander et al., 2001)
DNA
(Moroz et al., 2014; Prüfer
et al., 2014; Wang et al., 2013)
3
2
(Carroll, 2005)
Pax6
(Gehring and Ikeo,
1999)
François Jacob
1969
Roy J. Britten
(Britten and Davidson, 1969; Romero et al., 2012)
(Guenther et al., 2014;
Herrera et al., 2013; McLean et al., 2011; Sasaki et al., 2008)
DNA
4
Conrad Waddington
(Waddington, 1942)
Arthur D. Riggs
1996
DNA
Epigenetic Inheritance
/
(Bird, 2007; Russo
et al., 1996)
DNA
DNA
H1
H2A
2
H2B
H3
H4
8
H2A
H2B
H3
H4
DNA
(Cedar and Bergman, 2009)
4
(H3K4)
MLL
H3
27
SETD1
(H3K27)
ASH1L
(me1
me2
me3)
H3K4
3
(Ruthenburg et al., 2007)
H3K4me2
H3K4me1
H3K27
PRC2
5
EZH2
H3K4me3
(Bernstein et al., 2007)
H3K27
DNA
DNA
CpG
CpG
5
60~90%
(Bird, 2002)
X
3
DNA
DNA
(DNMT1
DNMT1
DNMT3A
DNMT3B)
DNA
DNMT3
DNA
TET
DNA
(Pastor
DNA
et al., 2013)
DNA
DNA
DNA
DNA
DNA
DNA
(Wu and Zhang, 2010)
6
TET
DNA
TET
(He et al., 2011; Ito et al., 2011)
DNA
DNA
DNA
DNA
Chromatin Immuno-Precipitation
sequencing (ChIP-seq)
Whole Genome Bisulfite Sequencing (WGBS)
(Ho et al., 2014; Pope et al., 2014)
7
RNA-seq
RNA
non-coding RNA (ncRNA)
ncRNA
(Carninci et al.,
ncRNA (lncRNA)
2005)
ncRNA
200 nt
ncRNA
(Imamura et
promoter-associated ncRNA (pancRNA)
al., 2004) (Figure 1)
ncRNA
pancRNA Khps1
Sphk1
pancRNA
Sphk1
Khps1
lncRNA
Khps1
Sphk1
(Imamura et al., 2004)
DNA
pancRNA
in vitro
DNA
Sphk1
pancRNA
Sphk1
Nefl
Esr1
Ar
Pgr
pancRNA
Vim
Gck
pancRNA
DNA
(Tomikawa et al., 2011) Vim
Nefl
pancRNA
8
A
TSS
B
TSS
mRNA
mRNA
pancRNA
Figure 1. pancRNA
DNA
pancRNA
(TSS)
CpG
(A) pancRNA
mRNA
pancRNA
(B) pancRNA
DNA
9
mRNA
pancRNA
pancRNA
lncRNA
pancRNA
pancRNA
(Carroll, 2008; Uesaka and Imamura, 2012)
pancRNA
pancRNA
pancRNA
1
pancRNA
2
pancRNA
ncRNA
pancRNA
pancRNA
10
第 1 章 一本鎖 lncRNA、pancRNA による遺伝子発現制御の普遍
性
序論
(Lander et al.,
1.5%
2001)
FANTOM
ENCODE
62%
ncRNA
(Carninci et al., 2005; The ENCODE Project Consortium, 2012)
ncRNA
micro RNA (miRNA)
interacting RNA (piRNA)
small RNA
RNA-RNA
RNA
(Moazed, 2009;
RNA
Nishi et al., 2013; Siomi and Siomi, 2009)
lncRNA
RNA-RNA
piwi-
small RNA
RNA
(Beltran et al., 2008; Carrieri et
al., 2012; Ebralidze et al., 2008; Hastings et al., 2000; Kimelman and Kirschner,
1989; Munroe and Lazar, 1991; Spigoni et al., 2010; Wagner et al., 1989)
RNA-RNA
4,520
RNA-RNA
sense-antisense transcript (SAT) pair
(Katayama et al., 2005)
ncRNA
mRNA
DNA
(Ambros, 2004; Matzke
11
and Birchler, 2005; Morris et al., 2004; Turner and Morris, 2010)
mRNA
ncRNA
RNA-RNA
mRNA
RNA-RNA
mRNA
HOTTIP
HOXA
lncRNA
5’
lncRNA
WD repeat-containing protein 5 (WDR5)
mixed-lineage
(Wang et al., 2011)
leukaemia (MLL) histone methyltransferase
HOTTIP-WDR5-MLL1
H3K4me3
DBE-T
4q35
lncRNA
group
1
ASH1L
(Cabianca et al., 2012)
ASH1L
DBE-T
DBE-T
Trithorax
4q35
H3K36
4q35
lncRNA
RNA-RNA
RNA-RNA
small RNA
RNA-RNA
lncRNA
12
CpG
CpG
(CGI)
(Antequera, 2003;
Lavia et al., 1987)
head-to-head (HtH)
mRNA
pancRNA
mRNA
(Imamura et al.,
2004; Tomikawa et al., 2011)
pancRNA
mRNA
HtH
pancRNA
mRNA
13
材料と方法
(Mus musculus; Japan SLC)
C57BL6
14
10
(5:00~19:00
)
16
-80
(Great Ape
Information Network; GAIN)
(Pan troglodytes)
28
BA10
-80
TRIzol
(Life Technologies)
total RNA
17.5
ICR
(Mus musculus; Japan SLC)
4.17 mM
NaHCO3
Ca2+
Mg2+
HBSS (Hank’s balanced solt solution; SIGMA)
0.33 g/l L-cystein (Nacalai
Tesque)
0.33 g/l BSA (SIGMA)
8.33 g/l glucose
3.3% papain from papaya latex (SIGMA)
37
20
500 U/ml DNase I (SIGMA)
Ca2+
0.6% glucose
14
Mg2+
5% FBS
1% Antibiotic-
PBS
Antimycotic Mixed Stock Solution (Nacalai Tesque)
MEM Alpha (Gibco)
poly-L-
lysine
MEM alpha
CO2
3
1% GlutaMax (Life Technologies)
37
2% B27 supplement (Life Technologies)
1% Antibiotic-Antimycotic Mixed Stock Solution
Neurobasal medium (Gibco)
37
3
CO2
10 µM
AraC (SIGMA)
shRNA
RNA
shRNA
pLLX-shRNA
C
GFP
shRNA
shRNA
Table 1
HEK293T
VSV-G
pLLX-shRNA
shRNA
5
RNA
15
pLLX-
Table 1. shRNA
Target
pancVwa5b2
pancPacsin1
pancSh3rf3
Sequence
TGATATGAACAAATACTAAAGATTCAAGAGATCTTTAGTATTTGTTCATATCTTTTTTGGAAC
TGCTGCTGCCTTGTATTCTAACTTCAAGAGAGTTAGAATACAAGGCAGCAGCTTTTTTGGAAC
TGCAGATTCTCCTAAGCCATGTTTCAAGAGAACATGGCTTAGGAGAATCTGCTTTTTTGGAAC
16
Directional RNA
Directional RNA
(Directional RNA-seq)
Illumina
cDNA
µg
total RNA
Sera-Mag Magnetic Oligo
(dT) Beads (Thermo Scientific)
RNA
5
polyA+ RNA
rRNA
2
2%
Bioanalyzer chip (Agilent)
buffer (Affymetrix)
Total RNA Pico
polyA+ RNA
94
Fragmentation
3
RNA
TAP (Nippongene)
PCI
RNA
3’
Antarctic phosphatase
5'
kinase (NEB)
RNA
MinElute kit (Qiagen)
(NEB)
T4 polynucleotide
RNeasy
T4 RNA ligase 2, truncated K277Q
4
v1.5 sRNA 3’ adaptor (Illumina)
RNA
RNA
5’
RNA ligase (Illumina)
SRA 5’ adaptor (Illumina)
20
T4
1
SRA 5’ adaptor
SuperScriptIII First-Strand Synthesis System (Life
Technologies)
cDNA
2
Phusion DNA polymerase (FInnzymes)
98
15
3
cDNA
30
98
12
72
17
10
10
60
30
72
PCR
AMPure XP (Beckman Coulter)
cDNA
)
1st
2
(100 bp
)
2nd
(50 bp
HiSeq 2000
Directional RNA-seq
FASTX-Toolkit
(
99%
)
20 nt
50 bp
3’
2nd
RNA
TopHat
(mm9)
(Trapnell et al., 2009)
(hg19)
TopHat
v2.0.4
18
1st
(Figure 2)
PCR
1
(Djebali et al., 2012)
Directional RNA-seq
MACS
(Zhang et al., 2008)
v1.4.1
--nonmodel
FDR
MACS
0.05
Ensembl Genes Database
(Flicek et al.,
1,000 bp
2011)
100 bp
Directional RNA-seq
RNA
1,000 bp
RNA
1,000 bp
Reads Per Kilobase per Million mapped
reads (RPKM)
ncRNA
2
19
HtH
A Chr16:27,439,951-27,453,748
B Chr7:133,970,753-133,973,864
Ccdc55
Prcc
Before
Before
After
After
C Chr3:87,673,360-87,676,342
2310061C15R
Before
After
Figure 2.
Directional RNA-seq
PCR
20
Cufflinks
(Trapnell et al., 2010)
RPKM
RNA
RNA
MACS
HtH
RNA
RNA
2,000 bp
(ORF)
ORF
EMBOSS
(Rice et al., 2000)
ORF
ORF
De novo
R
Bioconductor
rGADEM
2014; Gentleman et al., 2004)
CCGCCG
1,000 bp
(Droit et al.,
v1.0.1
CGGCGG
1,000 bp
100 bp
RT-PCR
RT-PCR
TRIzol
cDNA
(Life Techmologies)
total RNA
21
DNase I (Life
Technologies)
Oligo-dT
SuperScriptIII First-Strand Synthesis System (Life Technologies)
RT-PCR
KAPA SYBR Fast qPCR Kit (KAPA Biosystems)
Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies)
Gapdh
Table 2
Strand-specific RT-PCR
Table 3
22
Table 2.
Target
Vwa5b2
RT-PCR
Forward
Reverse
pancVwa5b2 Forward
Reverse
Pacsin1
Forward
Reverse
pancPacsin1 Forward
Reverse
Sh3rf3
Forward
Reverse
pancSh3rf3
Forward
Reverse
Gapdh
Forward
Reverse
Primer sequence
CCAGAGGAGGTGTTATCCGC
GTCAGAGCTTCCATACTCTGCT
GGGAAGTGAGCGAAGGTAAGT
AGTTCTGACTGCTCCCACCT
TGATGGTGTCCGGTGCTC
TTGTAGTTCCCCACCTCCCA
TGCATGCAGACGCTTGTATTG
TTAGTCAGGGAACCGAGGGA
CATCTGTCCTGCACTGTCCC
ACTTTGGGAGACATGCCCTG
CAGCCCTTAGCCTGTAGTCC
GAGAGGGTCCAGAATGCCTG
TCCACCACCCTGTTGCTGTA
ACCACAGTCCATGCCATCAC
23
Table 3.
Locus
Pacsin1
Kcnmb4
RT-PCR
Name
a
b
c
d
e
f
g
h
q
r
s
i
j
k
l
m
n
o
p
v
w
x
y
Primer sequence
CGATCTGATAAGCCCCTTCGTAA
AGCTGCTGCCTTGTATTCTAACT
ATCCCTCGGTTCCCTGACTAATA
CTTGAGAGACCTGGATGTTCACA
CTTCTCTCACCTCCGGATCTCTC
GGAGACTCTTTGGTGGACTCTAT
GGAGAAGTATGAGAAGGTGCTGG
AGATGTGACATTGGGTTAAGTCC
CGCTCCTAATACCTGCTTCTCAT
ATGAGAAGCAGGTATTAGGAGCG
CTTCCTCCTTCCCGCAGC
GTTTTTCCAAGTCCTGCAGCC
TGCGAAGCGGGAATCTTCAA
GCCAGCTAAGGGTGGCAATA
CGTGCAGGCTAACACGATTG
ACGTGAACAACTCCGAGTCC
CCAACTGTGCCTGTTTCTGC
GATCGGTTCCCAGCCATTCA
ACCACGATGAGAACACCCAC
GAGCTGCTCTCGGAATCCTC
AGGAGCAGCCTCGCTCAA
TTCGAGTGCACCTTCACCTG
GGAGTTGGACTCGGAGTTGTT
24
結果
方向と
方向の転写が同じ領域から起こる場合は非常に稀である
Directional
RNA-seq
1st
2nd
2
7.6 ± 0.1×10!
1st
2.3 ± 0.1×10!
2nd
(Table 4)
1st
0.1×10!
7.2 ±
2.0 ± 0.1×10!
2nd
Directional RNA-seq
(
86.1%
1
)
85.4%
78.1%
72.6%
82.0%
PCR
1.9×10!
1.8×10!
2.3×10!
3.0×10!
1.9×10!
(
)
25
26
220,364,813
Mouse Heart (n=2)
e
d
c
b
a
233,051,369
Mouse Heart (n=1)
76,687,574
Mouse Cerebral Cortex (n=2)
238,989,445
76,557,221
Mouse Cerebral Cortex (n=1)
Mouse Cerebellum (n=2)
76,081,033
Chimpanzee Cerebellum (n=2)
220,787,053
76,342,191
Chimpanzee Cerebellum (n=1)
2nd run Mouse Cerebellum (n=1)
77,141,984
Chimpanzee Cerebral Cortex (n=2)
(%)
b
191,584,172 86.9%
201,311,951 86.4%
207,347,311 86.8%
192,894,809 87.4%
72,437,179 94.5%
72,322,648 94.5%
71,963,443 94.6%
72,237,582 94.6%
72,913,619 94.5%
70,894,403 94.5%
Total # reads # Valid reads
75,012,641
Sample
a
c
(PCR
181,795,500 94.9%
190,937,364 94.8%
195,402,638 94.2%
181,763,145 94.2%
68,665,190 94.8%
68,635,440 94.9%
63,973,178 88.9%
64,283,120 89%
63,089,059 86.5%
61,404,334 86.6%
# Mapped reads (%)
Directional RNA-seq
1st run Chimpanzee Cerebral Cortex (n=1)
Table 4.
)
139,227,099 72.7%
146,153,387 72.6%
177,060,051 85.4%
164,728,033 85.4%
62,418,231 86.2%
62,286,305 86.1%
58,955,648 81.9%
59,273,070 82.1%
56,935,814 78.1%
55,436,357 78.2%
# Uniquely mapped reads (%)
d
17,875,501
18,243,843
30,571,226
30,097,533
19,814,914
18,552,890
22,396,483
22,562,341
19,057,212
18,894,958
after removal of duplication
# Uniquely mapped reads
1.01
1.01
1.03
1.03
1.02
1.02
1.04
1.04
1.03
1.03
/ Bottom strand-mapped readse
Top strand-mapped reads
(
)
1.0
(Table 4)
Directional RNA-seq
Pacsin1
pancPacsin1 Kcnmb4
Strand-specific RT-PCR
(Figure 3A, B)
pancKcnmb4
Strand-specific RT-PCR
Directional RNA-seq
Directional RNA-seq
RPKM
Kendall
0.96
(p < 0.001)
2
1
polyA+ RNA
25.0% 30.0% 21.6%
(Table 5)
polyA+ RNA
0.7%
1.3% 0.7%
(Table 5)
27
A
+1
Pacsin1 locus
5'
a
c
d
b
e
c-d
PCR primer
a
RT primer
RT rxn +
-
+
a
-
+
b
-
-
f
-
+
e
-
+
f
-
+
-
+1
5'
i
k
j
m
i
RT rxn +
-
+
i
-
+
j
-
+
Figure 3. pancRNA
m
n
-
+
-
Heart
m
n
+ - + -
Kcnmb4
Pacsin1 locus
PCR primer c - d
RT rxn + -
Cx
RT rxn +
-
3'
n
o-p
RT primer
pancKcnmb4
c
p
Samples
Heart
j
o
PCR primer
Cx
Samples
RT primer
l
k-l
PCR primer
5'
Heart
Pacsin1
Kcnmb4 locus
C
e
RT rxn +
pancPacsin1
B
Cx
RT primer
+
3'
f
g-h
Samples
Heart
b
h
PCR primer
Cx
Samples
g
D
+1
d
q r
c-q
+ -
s
h
r-h
+ -
s-h
+ -
Kcnmb4 locus
3'
5'
k
l
PCR primer k - l
RT rxn + -
+1
t u
k-t
+ -
v
w
u-w v-w
+ - + -
mRNA
Pacsin1 (A) (C)
Kcnmb4 (B) (D)
pancRNA
mRNA
+
(A) (B)
specific RT-PCR
(Cx)
(Heart)
(C) (D)
(Cx)
Strand-specific RT-PCR
28
Strand(Heart)
3'
Table 5.
Mouse Cerebral Cortex
Mouse Cerebellum
Mouse Heart
Chimpanzee Cerebral Cortex
Chimpanzee Cerebellum
Transcribed Unidirectionally
Bidirectionally
a
regions
transcribed regions transcribed regionsb
25.0%
24.3%
0.7%
30.0%
28.7%
1.3%
21.6%
20.9%
0.7%
24.5%
23.6%
0.9%
23.4%
22.2%
1.2%
a
b
29
(Figure 4)
RNA-RNA
pancRNA
HtH
ENCODE
GENCODE
48%
CAGE-seq
100 bp
(Djebali et al., 2012)
lncRNA
2
HtH
RNA
ORF
1,000 bp
1,000 bp
ORF
191.5
319.6
213.0
305.0
30
B
Mouse Cereberal Cortex
Mouse Cerebellum
0.35
0.35
0.30
0.30
0.25
0.25
Density
Density
A
0.20
0.15
0.20
0.15
0.10
0.10
0.05
0.05
0.00
0.00
10
-3
10
-2
10
-1
1
10
1
10
2
10
10-4
2
10-2
1
104
10
sense mapped read count
/ antisense mapped read count
3
sense mapped read count
/ antisense mapped read count
C
D
Mouse Heart
Chimpanzee Cerebral Cortex
0.30
0.35
0.25
0.30
0.25
Density
Density
0.20
0.15
0.10
0.20
0.15
0.10
0.05
0.05
0.00
0.00
10-4
10-2
1
102
104
sense mapped read count
/ antisense mapped read count
E
-2
Chimpanzee Cerebellum
0.35
0.30
Density
0.25
0.20
0.15
0.10
0.05
0.00
10-4
2
10
1
10
sense mapped read count
/ antisense mapped read count
10-2
1
102
104
sense mapped read count
/ antisense mapped read count
Figure 4.
DNA
31
ORF
900 nt
(Figure 5)
ORF
NCBI
(Marchler-Bauer et al., 2011)
Conserved Domain Database
1,000 bp
Conserved Protein Domain
1.9%
bp
4.8%
1,000
Conserved Protein Domain
20.3%
15.5%
ncRNA
RNA
RNA
pancRNA
mRNA
(Figure 6; Table 6)
pancRNA
100
100
1,000 bp
RPKM
pancRNA
RPKM
32
A
Mouse Cerebral Cortex
Sequences of the downstream regions
0.006
0.006
0.005
0.005
0.004
0.004
Density
Density
Reverse complementary sequences of
the upstream regions
0.003
0.003
0.002
0.002
0.001
0.001
0.000
0.000
0
200
400
600
Length (nt)
800
1000
0
200
400
600
Length (nt)
800
1000
B
Chimpanzee Cerebral Cortex
Sequences of the downstream regions
0.006
0.006
0.005
0.005
0.004
0.004
Density
Density
Reverse complementary sequences of
the upstream regions
0.003
0.003
0.002
0.002
0.001
0.001
0.000
0.000
0
200
400
600
Length (nt)
800
1000
0
Figure 5.
200
400
600
Length (nt)
ORF
33
800
1000
Mouse Cerebral Cortex
B
pancRNAs
with the 100 top-ranked RPKM
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0
500 1000
-1000 -500
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Antisense mapped
read count
Sense mapped
read count
Antisense mapped
read count
1.6
mRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
mRNAs
with the 100 top-ranked RPKM
D
Sense mapped
read count
0.6
0.8
sense
antisense
1.6
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Figure 6.
34
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
pancRNAs
with the 100 top-ranked RPKM
(Group III)
Antisense mapped
read count
Sense mapped
read count
0.8
0.8
-1000 -500
0
500 1000
Position relative to TSS
pancRNAs
with the 100 top-ranked RPKM
(Group II)
pancRNAs
with the 100 top-ranked RPKM
(Group I)
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
C
pancRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
0.6
Sense mapped
read count
sense
antisense
0.8
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
Total Genes
Antisense mapped
read count
A
0.8
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
(A) (B) (C) (D)
(E) (F) (G) (H)
(K) (L)
(I) (J)
(M) (N) (O) (P)
(S) (T)
(Q) (R)
(A) (E) (I) (M) (Q)
100
pancRNA
pancRNA
(
)
(
(B) (F) (J) (N) (R)
)
100
(C) (G) (K) (O) (S)
3
100
pancRNA
I
1,000 bp
1,000 bp
5
(Group I;
)
II
1,000 bp
1,000 bp
2
I
100
(Group II;
II
(Group III;
(
)
100
35
)
)
III
(D) (H) (L) (P) (T)
(
)
Mouse Cerebellum
F
pancRNAs
with the 100 top-ranked RPKM
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0
500 1000
-1000 -500
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Antisense mapped
read count
Sense mapped
read count
Antisense mapped
read count
1.6
mRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
mRNAs
with the 100 top-ranked RPKM
H
Sense mapped
read count
0.6
0.8
sense
antisense
1.6
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Figure 6. continued
36
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
pancRNAs
with the 100 top-ranked RPKM
(Group III)
Antisense mapped
read count
Sense mapped
read count
0.8
0.8
-1000 -500
0
500 1000
Position relative to TSS
pancRNAs
with the 100 top-ranked RPKM
(Group II)
pancRNAs
with the 100 top-ranked RPKM
(Group I)
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
G
pancRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
sense
antisense
Sense mapped
read count
0.8
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
Total Genes
Antisense mapped
read count
E
0.8
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
Mouse Heart
J
pancRNAs
with the 100 top-ranked RPKM
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0
500 1000
-1000 -500
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Antisense mapped
read count
Sense mapped
read count
Antisense mapped
read count
1.6
mRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
mRNAs
with the 100 top-ranked RPKM
L
Sense mapped
read count
0.6
0.8
sense
antisense
1.6
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Figure 6. continued
37
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
pancRNAs
with the 100 top-ranked RPKM
(Group III)
Antisense mapped
read count
Sense mapped
read count
0.8
0.8
-1000 -500
0
500 1000
Position relative to TSS
pancRNAs
with the 100 top-ranked RPKM
(Group II)
pancRNAs
with the 100 top-ranked RPKM
(Group I)
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
K
pancRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
sense
antisense
Sense mapped
read count
0.8
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
Total Genes
Antisense mapped
read count
I
0.8
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
Chimpanzee Cerebral Cortex
N
pancRNAs
with the 100 top-ranked RPKM
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0
500 1000
-1000 -500
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Antisense mapped
read count
Sense mapped
read count
Antisense mapped
read count
1.6
mRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
mRNAs
with the 100 top-ranked RPKM
P
Sense mapped
read count
0.6
0.8
sense
antisense
1.6
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Figure 6. continued
38
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
pancRNAs
with the 100 top-ranked RPKM
(Group III)
Antisense mapped
read count
Sense mapped
read count
0.8
0.8
-1000 -500
0
500 1000
Position relative to TSS
pancRNAs
with the 100 top-ranked RPKM
(Group II)
pancRNAs
with the 100 top-ranked RPKM
(Group I)
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
O
pancRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
sense
antisense
Sense mapped
read count
0.8
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
Total Genes
Antisense mapped
read count
M
0.8
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
Chimpanzee Cerebellum
R
pancRNAs
with the 100 top-ranked RPKM
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0
500 1000
-1000 -500
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
0.4
0.2
0.0
0.2
0.4
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
-1000 -500
0
500 1000
Position relative to TSS
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Antisense mapped
read count
Sense mapped
read count
Antisense mapped
read count
1.6
mRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
mRNAs
with the 100 top-ranked RPKM
T
Sense mapped
read count
0.6
0.8
sense
antisense
1.6
1.2
0.8
0.4
0.0
0.4
0.8
1.2
1.6
-1000 -500
0
500 1000
Position relative to TSS
Figure 6. continued
39
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
pancRNAs
with the 100 top-ranked RPKM
(Group III)
Antisense mapped
read count
Sense mapped
read count
0.8
0.8
-1000 -500
0
500 1000
Position relative to TSS
pancRNAs
with the 100 top-ranked RPKM
(Group II)
pancRNAs
with the 100 top-ranked RPKM
(Group I)
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
S
pancRNAs
with the 100 bottom-ranked RPKM
Sense mapped
read count
sense
antisense
Sense mapped
read count
0.8
Antisense mapped
read count
Antisense mapped
read count
Sense mapped
read count
Total Genes
Antisense mapped
read count
Q
0.8
sense
antisense
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
-1000 -500
0
500 1000
Position relative to TSS
Table 6.
TSS
RPKM
Upstream region
Downstream region
Sample
Subgroup
Antisense RPKM
Sense RPKM
Antisense RPKM
Sense RPKM
Mouse
Cerebral Cortex
Total genes
Top 100 ranked antisense RPKM located upstreama
15.1
734.3
10.7
9.1
4.9
195.6
145
267.5
& Low antisense RPKM located downstreamb
& Middle antisense RPKM located downstreamb
427.5
165
b
& High antisense RPKM located downstream
Mouse
Cerebellum
141.8
Bottom 100 ranked antisense RPKM located upstreamc
Top 100 ranked sense RPKM located downstreamd
Bottom 100 ranked sense RPKM located downstreame
Total genes
Top 100 ranked antisense RPKM located upstreama
0
26.4
3.3
19.4
790.7
& Low antisense RPKM located downstreamb
& Middle antisense RPKM located downstreamb
0
50.1
2.1
15.7
848.6
& Low antisense RPKM located downstreamb
& Middle antisense RPKM located downstreamb
579
120.7
& High antisense RPKM located downstreamb
Chimpanzee
Cerebral Cortex
0
70.4
2.0
16.2
653.6
& Low antisense RPKM located downstreamb
& Middle antisense RPKM located downstreamb
401.4
147.7
& High antisense RPKM located downstreamb
Chimpanzee
Cerebellum
0
25.3
5.4
16.1
649.9
& Low antisense RPKM located downstreamb
& Middle antisense RPKM located downstreamb
402.1
85
& High antisense RPKM located downstreamb
0
59.9
4.4
a TSS
RPKM
100
b TSS
RPKM
100
1,000 bp
(Low antisense RPKM in downstream region)
40
131.2
22.8
158.6
0.5
44.8
4.8
1,000 bp
5
40.4
78.4
1636.6
0
102.4
214.5
27.9
24.7
1.6
9.3
119.2
0.6
146.6
44
82.8
0.9
14.5
5.1
6.3
211.2
24.2
0.7
162.8
Bottom 100 ranked antisense RPKM located upstreamc
Top 100 ranked sense RPKM located downstreamd
Bottom 100 ranked sense RPKM located downstreame
113
121.6
2542.2
0
114.3
231
18.7
48.5
0.5
9.5
94.9
1.0
10.7
26.6
246.8
129.3
137.9
0.5
40.9
1.6
5.9
150
5.1
1.7
104.4
Bottom 100 ranked antisense RPKM located upstreamc
Top 100 ranked sense RPKM located downstreamd
Bottom 100 ranked sense RPKM located downstreame
Total genes
Top 100 ranked antisense RPKM located upstreama
39.8
88.1
1736.3
0
211.2
489.1
27.9
41.5
3.1
8.9
181.6
0.3
10
7.3
206.1
124.6
115.3
0.5
22.7
1.9
6.5
207.2
15.2
1.1
148.9
Bottom 100 ranked antisense RPKM located upstreamc
Top 100 ranked sense RPKM located downstreamd
Bottom 100 ranked sense RPKM located downstreame
Total genes
Top 100 ranked antisense RPKM located upstreama
11.1
88.0
2054.4
0
174.8
370.5
32.3
65.6
0.6
8.4
236.3
0.3
10.5
19.4
181.5
74.9
116.4
0.4
7.3
2.9
8.1
213.2
7.4
7.3
137.3
Bottom 100 ranked antisense RPKM located upstreamc
Top 100 ranked sense RPKM located downstreamd
Bottom 100 ranked sense RPKM located downstreame
Total genes
Top 100 ranked antisense RPKM located upstreama
22.8
56.4
1.0
9.0
278.4
0.3
11.3
15.2
463
190.4
& High antisense RPKM located downstreamb
Mouse Heart
5.9
2.2
60.4
65.1
1484.9
0
3
1,000 bp
1,000 bp
2
(High antisense RPKM in downstream region)
(Middle antisense RPKM in downstream region)
c TSS
RPKM
100
d TSS
RPKM
100
e TSS
RPKM
100
41
2
pancRNA
pancRNA
100
100
(Figure 6A, B, E, F, I, J)
RPKM
pancRNA
mRNA
Figure 7)
(p < 0.001;
pancRNA
100
I
3
1,000 bp
1,000 bp
5
II
1,000 bp
1,000 bp
2
III
(Figure 6C, G, K)
I
II
pancRNA
mRNA
5’
Pacsin1
pancRNA
Kcnmb4
pancRNA
mRNA
HtH
(Figure 3C, D)
RNA-RNA
RNA
mRNA
100
pancRNA
mRNA
100
pancRNA
(Figure 6D, H, L)
42
B
5.0
Relative Average of RPKM
Relative Average of RPKM
A
4.0
3.0
2.0
1.0
***
0
Figure 7.
100
2.5
2.0
pancRNAs with
the 100 top-ranked RPKM
1.5
pancRNAs with
the 100 bottom-ranked RPKM
1.0
***
0.5
0
pancRNA
pancRNA
RPKM
(A)
mRNA
100
1,000 bp
RPKM
RPKM
p < 0.01;
Student
43
t
(B)
pancRNA
pancRNA
mRNA
mRNA
pancRNA
(Figure 6M-T;
Table 6)
pancRNA
mRNA
pancRNA
mRNA
(Imamura et al., 2001; 2004)
pancRNA
RPKM
pancRNA
pancRNA
pancRNA
mRNA
(Figure 8A; Table 7)
pancRNA
(Figure 8B; Table 7)
pancRNA
mRNA
pancRNA
mRNA
pancRNA
mRNA
44
Figure 8.
pancRNA
pancRNA
(A)
pancRNA
(B)
pancRNA
pancRNA
(C)
(D)
45
Table 7.
pancRNA
TSS
RPKM
Upstream region
Downstream region
Antisense RPKM Sense RPKM Antisense RPKM Sense RPKM
Cerebral Cortex vs Heart
(mouse)
Cerebral Cortex-specific
pancRNA-bearing genes
Heart-specific
pancRNA-bearing genes
Cerebellum vs Heart
(mouse)
Cerebellum-specific
pancRNA-bearing genes
Heart-specific
pancRNA-bearing genes
Cerebral Cortex vs Cerebellum
(mouse)
Cerebral Cortex-specific
pancRNA-bearing genes
Cerebellum-specific
pancRNA-bearing genes
Cerebral Cortex
98.1
18.5
12.9
369.9
Heart
2.2
4.6
2.9
163.8
Cerebral Cortex
3.7
12.1
1.4
172.3
Heart
83.7
18.6
6.4
516.6
Cerebellum
96.5
29.2
14.8
410.6
Heart
2.5
11.1
2.1
192.2
Cerebellum
5.2
9.1
3.7
203.3
Heart
144.8
16.7
7.7
486.1
Cerebral Cortex
74.6
16.1
7.8
304.3
Cerebellum
4.9
15.1
5.3
201.5
Cerebral Cortex
4.5
16
1.3
220.8
Cerebellum
70.5
21.8
7.8
302.6
pancRNA
RPKM
RPKM
0.3
0.1
46
mRNA
pancRNA
mRNA
(Figure 8C, D; Table 7)
pancRNA
mRNA
Vwa5b2
pancRNA
Pacsin1
pancRNA
17.5
pancRNA
pancRNA
PCR
mRNA
RT-
pancRNA
mRNA
pancRNA
(Figure 9)
mRNA
pancRNA
mRNA
NCBI Gene Expression Omnibus
19
2
Directional RNA-seq
pancRNA
pancRNA
mRNA
(Shen et al., 2012)
Pearson
pancRNA
pancRNA
mRNA
ncRNA
mRNA
47
0.93
HtH
0.81
.0
.8
.6
**
.4
.2
0
mRNA
Relative Expression Level
.2
Pacsin1 locus
1.2
1.2
1.0
1.0
0.8
0.8
**
0.6
0.4
0.2
0.2
pancRNA
**
0.6
0.4
0
Relative Expression Level
Vwa5b2 locus
0
1.2
1.2
1.0
1.0
0.8
0.8
0.6
0.6
**
0.4
0
Figure 9.
pancRNA knockdown
0.4
0.2
0.2
mRNA
Control
*
pancRNA
0
mRNA
pancRNA
y
RT-PCR
mRNA
RNA
Gapdh
RT-PCR
p < 0.01
p < 0.01;
48
Student
t
pancRNA
pancRNA
CGI (CpG
)
pancRNA
pancRNA
pancRNA
92.8%
91.4%
92.3%
CGI
pancRNA
94.7%
pancRNA
93.4%
CGI
pancRNA
CGI
CGI
pancRNA
de novo
HtH
pancRNA
CCG
(Figure 10A, C, E)
CGG
(Figure 10G, I)
CCG
(Figure 10G-J)
CCGCCG
1,000 bp
CGG
CCG
CGG
CGGCGG
1,000 bp
pancRNA
49
Mouse Cerebral Cortex
A
-100bp ~ +100bp
2
1.5
Bits
1.5
Bits
+300bp ~ +400bp
2
1
1
0.5
0.5
0
0
1
B
5
10
15
20
1
5
10
0.30
0.25
0.25
0.20
0.20
0.15
CCGCCG
CGGCGG
0.15
0.10
0.10
0.05
0.05
0.00
-1000
Figure 10. pancRNA
CCGCCG
CGGCGG
0.00
-1000
-500
0
500
1000
Position relative to TSS
-500
0
500
1000
Position relative to TSS
DNA
(A) (C) (E) (G) (I)
(
)
CCGCCG
(D)
20
HtH Promoter
0.30
Frequency
Frequency
Total Genes
15
(B) (D) (F) (H) (J)
pancRNA
(
CGGCGG
)
(A) (B)
(E) (F)
(G) (H)
(I) (J)
50
(C)
Mouse Cerebellum
C
-100bp ~ +100bp
2
1.5
Bits
1
1
0.5
0.5
0
0
1
D
5
10
1
15
5
Total Genes
0.30
0.25
0.25
0.20
0.20
0.15
CCGCCG
CGGCGG
0.15
0.10
0.10
0.05
0.05
0.00
-1000
10
15
20
HtH Promoter
0.30
Frequency
Bits
1.5
Frequency
+200bp ~ +300bp
2
0.00
-1000
-500
0
500
1000
Position relative to TSS
Figure 10. continued
51
CCGCCG
CGGCGG
-500
0
500
1000
Position relative to TSS
Mouse Heart
E
-100bp ~ +100bp
2
1.5
Bits
1
1
0.5
0.5
0
0
1
F
5
10
1
15
5
Total Genes
0.30
0.25
0.25
0.20
0.20
0.15
CCGCCG
CGGCGG
0.15
0.10
0.10
0.05
0.05
0.00
-1000
10
15
20
HtH Promoter
0.30
Frequency
Bits
1.5
Frequency
+100bp ~ +200bp
2
0.00
-1000
-500
0
500
1000
Position relative to TSS
Figure 10. continued
52
CCGCCG
CGGCGG
-500
0
500
1000
Position relative to TSS
Chimpanzee Cerebral Cortex
G
-100bp ~ +100bp
2
1.5
Bits
1
1
0.5
0.5
0
0
1
H
5
10
15
20
1
5
Total Genes
0.30
0.25
0.25
0.20
0.20
0.15
CCGCCG
CGGCGG
0.15
0.10
0.10
0.05
0.05
0.00
-1000
10
15
20
HtH Promoter
0.30
Frequency
Bits
1.5
Frequency
+400bp ~ +600bp
2
0.00
-1000
-500
0
500
1000
Position relative to TSS
Figure 10. continued
53
CCGCCG
CGGCGG
-500
0
500
1000
Position relative to TSS
Chimpanzee Cerebellum
I
+1bp ~ +200bp
2
1.5
Bits
1
1
0.5
0.5
0
0
1
J
5
10
15
20
1
5
Total Genes
0.30
0.30
0.25
0.25
0.20
0.20
0.15
CCGCCG
CGGCGG
0.15
0.10
0.10
0.05
0.05
0.00
-1000
10
15
20
HtH Promoter
Frequency
Bits
1.5
Frequency
+100bp ~ +300bp
2
0.00
-1000
-500
0
500
1000
Position relative to TSS
Figure 10. continued
54
CCGCCG
CGGCGG
-500
0
500
1000
Position relative to TSS
pancRNA
CGI
(Figure 10B, D, F, H, J)
CCGCCG
CGGCGG
pancRNA
CCGCCG
CGGCGG
pancRNA
47.8%
pancRNA
pancRNA
47.3%
47.5%
19.7%
pancRNA
61.2%
pancRNA
60.9%
36.8%
HtH
GC
CCG
55
CGG
考察
Directional RNA-seq
Directional RNA-seq
81.4%
80.1%
94.7%
87.8%
ENCODE
(Djebali et al., 2012)
88.7%
72.4%
78.2%
Ensembl Genes
19,895
197,870
RNA
Directional RNA-seq
25.0%
56
Directional RNA-seq
24.5%
ENCODE
Directional RNA-seq
20%
(Djebali et al., 2012)
Directional RNA-seq
lncRNA
ncRNA
RNA
ncRNA
(Beltran
RNA-RNA
et al., 2008; Carrieri et al., 2012; Hastings et al., 2000; Kimelman and Kirschner,
1989; Munroe and Lazar, 1991; Wagner et al., 1989)
ncRNA
mRNA
(Ebralidze et al., 2008; Spigoni et al., 2010)
(Wang et al.,
ncRNA
2011)
0.7% 1.4% 0.7%
ncRNA
57
pancRNA
ncRNA
lncRNA
mRNA
pancRNA
lncRNA
mRNA
2
1
mRNA
lncRNA
lncRNA
mRNA
mRNA
(Beltran et al., 2008; Ebralidze et al., 2008; Hastings et al., 2000; Spigoni et al.,
2010)
PU.1
mRNA
(Ebralidze et al., 2008)
ncRNA
2
mRNA
lncRNA
pancRNA
pancRNA
mRNA
pancRNA
mRNA
RNA-RNA
(Imamura et al., 2004; Tomikawa et al., 2011) Sphk1
pancRNA Khps1
Sphk1
Sphk1
CpG
(Imamura et al., 2004)
pancRNA
pancRNA
58
mRNA
(Figure 9)
pancRNA
mRNA
pancRNA
mRNA
pancRNA
mRNA
pancRNA
mRNA
mRNA
pancRNA
mRNA
pancRNA
pancRNA
pancRNA
mRNA
mRNA
pancRNA
pancRNA
lncRNA
3
HOTTIP
lncRNA
RNA-RNA
(Wang et al., 2011)
2
SAT
(Katayama et al., 2005)
lncRNA
pancRNA
59
3
pancRNA
CCG/CGG
pancRNA
pancRNA
90%
CGI
pancRNA
CCG
pancRNA
CpG
CGG
DNA
pancRNA
CpG
pancRNA
DNA
GC
CGI
3
CGI
pancRNA
pancRNA
CCG
CGG
pancRNA
R
DNA-RNA-DNA
R
(Ginno
DNA
et al., 2013; 2012)
pancRNA
CCG
DNA
pancRNA
R
R
60
CGG
pancRNA
CCG
pancRNA
CGG
DNA
pancRNA
pancRNA
ncRNA
pancRNA
61
第 2 章 pancRNA を介した遺伝子発現制御の種間多様性
序論
(Wray, 2007)
(Carroll, 2008)
Hox
KITLG (KIT Ligand
)
AR (Androgen receptor
)
62
(Guenther et al., 2014; McLean et al., 2011)
cDNA
mRNA
ncRNA
RNA
1
(Carninci et al., 2005)
ncRNA
RNA
pancRNA
Xist
miRNA
ncRNA
ncRNA
10
ncRNA
lncRNA
(Baker, 2011)
lncRNA
ncRNA
DNA
(Carroll, 2005)
lncRNA
(Engström et al., 2006;
Kutter et al., 2012; Necsulea et al., 2014)
lncRNA
2
63
lncRNA
lncRNA
ncRNA
(Khaitovich et al., 2005)
(Metzker, 2010)
DNA
RNA-seq
RNA
(Wang et al., 2009)
mRNA isoform
ncRNA
pancRNA
1
pancRNA
(Imamura et al., 2004; Tomikawa et al., 2011; Uesaka et
al., 2014)
pancRNA
pancRNA
pancRNA
64
pancRNA
65
材料と方法
(Great Ape Information Network; GAIN)
(
(Macaca mulatta)
)1
(Pan troglodytes)
-80
TRIzol
RNA
28
total RNA
(Life Technologies)
total
1
Directional RNA-seq
Directional
RNA-seq
Directional RNA-seq
Directional RNA-seq
Table 8
1
66
67
b
a
101
101
101
Peng et al., 2014
Peng et al., 2014
Peng et al., 2014
Kidney (n=2)
Liver (n=1)
Liver (n=2)
50
50
101
This manuscript
Heart (n=2)
50
Peng et al., 2014
This manuscript
Heart (n=1)
100
Kidney (n=1)
This manuscript
Cerebellum (n=2)
100
50
This manuscript
Cerebellum (n=1)
50
This manuscript
This manuscript
Cerebral Cortex (n=4)
76
Heart (n=4)
This manuscript
Cerebral Cortex (n=3)
100
This manuscript
This manuscript
Cerebral Cortex (n=2)
100
Heart (n=3)
This manuscript
Reference Read Length (nt)
Cerebral Cortex (n=1)
Chimpanzee Samples
26,736,656
25,050,505
47,285,536
69,204,269
172,129,601
153,616,605
172,682,050
182,261,526
76,081,033
76,342,191
198,660,786
150,132,652
77,141,984
75,012,641
# Raw readsa
24,779,948
23,238,657
43,925,321
63,801,245
159,985,054
143,146,155
159,865,447
168,421,525
72,404,439
72,412,322
185,044,719
140,684,461
73,114,615
71,196,725
# Valid readsb
Table 8. 第 2 章における Directional RNA-seq データの概要
92.7%
92.8%
92.9%
92.2%
92.9%
93.2%
92.6%
92.4%
95.2%
94.9%
93.1%
93.7%
94.8%
94.9%
(%)c
20,744,093
19,640,598
38,143,827
55,073,435
148,860,939
132,769,168
148,910,355
156,252,434
64,178,888
64,107,107
170,686,551
125,404,297
64,262,655
62,660,605
# Mapped readsd
83.7%
84.5%
86.8%
86.3%
93.0%
92.8%
93.1%
92.8%
88.6%
88.5%
92.2%
89.1%
87.9%
88.0%
(%)e
19,007,358
17,979,578
35,213,809
50,737,584
119,900,136
106,635,802
116,363,203
121,490,883
59,560,160
59,594,686
150,481,677
113,505,310
58,439,377
57,036,267
#Uniquely mapped
readsf
76.7%
77.4%
80.2%
79.5%
74.9%
74.5%
72.8%
72.1%
82.3%
82.3%
81.3%
80.7%
79.9%
80.1%
(%)g
95.8%
95.6%
96.5%
96.9%
96.7%
97.0%
97.3%
97.3%
93.3%
93.2%
89.8%
92.6%
96.8%
96.7%
Strandnessh
68
h
g
f
e
d
c
69
Merkin et al., 2012
Merkin et al., 2012
Liver (n=3)
Merkin et al., 2012
Kidney (n=1)
Liver (n=2)
Merkin et al., 2012
Heart (n=3)
Merkin et al., 2012
Merkin et al., 2012
Heart (n=2)
Liver (n=1)
Merkin et al., 2012
Heart (n=1)
Merkin et al., 2012
This manuscript
Cerebellum (n=2)
Kidney (n=3)
This manuscript
Cerebellum (n=1)
Merkin et al., 2012
This manuscript
Cerebral Cortex (n=2)
Kidney (n=2)
This manuscript
40
80
40
40
80
40
40
80
40
50
50
50
100
Reference Read Length (nt)
Cerebral Cortex (n=1)
Macaque Samples
Table 8. continued
26,700,682
113,094,939
28,555,788
40,389,069
108,637,672
31,891,747
36,101,619
109,193,093
35,248,042
241,173,391
232,575,935
272,056,320
76,074,284
# Raw readsa
22,839,833
104,559,333
24,426,241
35,291,681
100,348,848
27,527,410
31,669,553
99,169,204
29,370,416
208,583,939
201,001,979
227,840,845
72,341,454
# Valid readsb
85.5%
92.5%
85.5%
87.4%
92.4%
86.3%
87.7%
90.8%
83.3%
86.5%
86.4%
83.7%
95.1%
(%)c
19,216,226
95,061,998
20,620,967
30,623,422
90,028,805
23,835,660
28,251,878
90,299,167
26,329,604
189,016,875
182,131,497
201,824,544
59,951,525
# Mapped readsd
84.1%
90.9%
84.4%
86.8%
89.7%
86.6%
89.2%
91.1%
89.6%
90.6%
90.6%
88.6%
82.9%
(%)e
16,875,095
85,848,577
18,091,129
26,956,916
81,025,752
19,542,632
22,771,079
79,574,651
21,023,351
171,334,356
165,144,558
174,613,505
51,971,253
#Uniquely mapped
readsf
73.9%
82.1%
74.1%
76.4%
80.7%
71.0%
71.9%
80.2%
71.6%
82.1%
82.2%
76.6%
71.8%
(%)g
94.7%
98.1%
93.1%
97.7%
98.9%
98.4%
98.5%
98.9%
97.6%
96.1%
96.2%
97.8%
98.8%
Strandnessh
70
Cortez et al., 2014
Cortez et al., 2014
Cortez et al., 2014
Cortez et al., 2014
Heart (n=1)
Heart (n=2)
Kidney (n=1)
Kidney (n=2)
Cortez et al., 2014
Cortez et al., 2014
Cerebellum (n=2)
Liver (n=2)
Cortez et al., 2014
Cerebellum (n=1)
Cortez et al., 2014
Cortez et al., 2014
Cerebral Cortex (n=2)
Liver (n=1)
100
Cortez et al., 2014
Cerebral Cortex (n=1)
100
100
100
100
100
100
100
100
100
Reference Read Length (nt)
Marmoset Samples
Table 8. continued
47,504,110
32,192,774
24,017,790
24,553,329
30,211,556
24,859,231
29,474,916
38,437,551
26,093,403
36,544,979
# Raw readsa
43,840,256
29,934,177
22,068,454
22,835,038
27,507,700
22,993,386
27,127,725
35,742,273
24,080,926
34,049,298
# Valid readsb
92.3%
93.0%
91.9%
93.0%
91.1%
92.5%
92.0%
93.0%
92.3%
93.2%
(%)c
38,591,315
24,204,841
18,441,811
18,867,572
19,756,929
10,979,550
22,878,776
29,873,766
20,450,593
27,997,099
# Mapped readsd
88.0%
80.9%
83.6%
82.6%
71.8%
47.8%
84.3%
83.6%
84.9%
82.2%
(%)e
36,012,068
22,687,447
16,835,897
17,382,297
17,388,050
9,822,862
21,744,037
28,410,481
19,165,202
26,505,853
#Uniquely mapped
readsf
82.1%
75.8%
76.3%
76.1%
63.2%
42.7%
80.2%
79.5%
79.6%
77.8%
(%)g
98.0%
97.7%
97.9%
97.7%
98.2%
98.4%
97.0%
97.1%
97.8%
98.7%
Strandnessh
71
This manuscript
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Heart (n=3)
Heart (n=4)
Heart (n=5)
Heart (n=6)
Kidney (n=1)
Kidney (n=2)
Kidney (n=3)
This manuscript
Cerebellum (n=2)
Heart (n=2)
This manuscript
Cerebellum (n=1)
This manuscript
Lin et al., 2014
Cerebral Cortex (n=6)
Heart (n=1)
Lin et al., 2014
Cerebral Cortex (n=5)
Lin et al., 2014
This manuscript
Cerebral Cortex (n=4)
Cerebellum (n=4)
This manuscript
Cerebral Cortex (n=3)
Lin et al., 2014
This manuscript
Cerebral Cortex (n=2)
Cerebellum (n=3)
This manuscript
76
76
76
76
76
76
76
50
50
100
100
50
50
100
100
50
50
100
100
Reference Read Length (nt)
Cerebral Cortex (n=1)
Mouse Samples
Table 8. continued
18,788,286
35,536,674
38,866,777
39,528,591
41,443,334
35,570,309
39,038,956
220,364,813
233,051,369
144,705,041
150,974,801
238,989,445
220,787,053
166,188,101
155,680,801
163,175,074
190,260,665
76,687,574
76,557,221
# Raw readsa
17,593,099
30,938,735
35,914,919
36,550,245
37,095,579
32,289,619
35,744,773
191,584,172
201,311,951
130,861,596
132,292,186
207,347,311
192,894,809
150,895,675
141,873,780
152,258,703
176,888,419
72,678,857
72,746,201
# Valid readsb
93.6%
87.1%
92.4%
92.5%
89.5%
90.8%
91.6%
86.9%
86.4%
90.4%
87.6%
86.8%
87.4%
90.8%
91.1%
93.3%
93.0%
94.8%
95.0%
(%)c
15,529,202
27,067,678
32,299,831
33,135,980
33,498,158
29,860,544
31,789,795
181,603,560
190,758,663
115,989,016
118,402,104
195,296,960
181,665,861
132,383,301
127,236,073
142,266,235
165,286,797
63,976,355
66,724,119
# Mapped readsd
88.3%
87.5%
89.9%
90.7%
90.3%
92.5%
88.9%
94.8%
94.8%
88.6%
89.5%
94.2%
94.2%
87.7%
89.7%
93.4%
93.4%
88.0%
91.7%
(%)e
13,917,877
23,919,090
28,323,094
25,432,767
25,957,233
22,419,330
24,561,362
137,960,243
144,938,073
106,370,671
108,717,401
175,845,744
163,580,348
117,518,324
115,162,949
128,385,369
149,117,432
58,706,145
61,100,809
#Uniquely mapped
readsf
79.1%
77.3%
78.9%
69.6%
70.0%
69.4%
68.7%
72.0%
72.0%
81.3%
82.2%
84.8%
84.8%
77.9%
81.2%
84.3%
84.3%
80.8%
84.0%
(%)g
98.3%
98.1%
97.9%
98.7%
98.8%
98.9%
98.9%
99.3%
99.2%
98.3%
98.1%
99.2%
98.8%
98.2%
98.1%
99.2%
99.0%
99.3%
99.2%
Strandnessh
72
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Lin et al., 2014
Kidney (n=4)
Kidney (n=5)
Kidney (n=6)
Liver (n=1)
Liver (n=2)
Liver (n=3)
Liver (n=4)
Liver (n=5)
Liver (n=6)
76
76
76
76
76
76
76
76
76
30,633,616
24,234,923
27,549,415
31,913,472
17,894,796
30,461,949
39,936,163
37,408,520
40,542,680
28,280,772
22,640,190
25,736,055
28,387,634
16,716,090
28,393,959
37,392,167
34,593,130
37,987,761
92.3%
93.4%
93.4%
89.0%
93.4%
93.2%
93.6%
92.5%
93.7%
25,566,780
20,512,191
23,322,751
25,341,851
14,865,944
25,386,926
33,458,636
30,951,511
33,682,613
90.4%
90.6%
90.6%
89.3%
88.9%
89.4%
89.5%
89.5%
88.7%
22,038,962
17,822,001
20,147,045
21,750,682
12,922,577
21,868,110
28,460,726
26,019,856
28,461,665
77.9%
78.7%
78.3%
76.6%
77.3%
77.0%
76.1%
75.2%
74.9%
97.4%
97.7%
97.5%
97.9%
98.4%
97.9%
97.1%
97.3%
97.1%
73
Cortez et al., 2014
Cortez et al., 2014
Cortez et al., 2014
Cortez et al., 2014
Heart (n=1)
Heart (n=2)
Kidney (n=1)
Kidney (n=2)
Cortez et al., 2014
Cortez et al., 2014
Cerebellum (n=2)
Liver (n=2)
Cortez et al., 2014
Cerebellum (n=1)
Cortez et al., 2014
Cortez et al., 2014
Cerebral Cortex (n=2)
Liver (n=1)
100
Cortez et al., 2014
Cerebral Cortex (n=1)
100
100
100
100
100
100
100
100
100
Reference Read Length (nt)
Rat Samples
Table 8. continued
26,289,552
23,597,799
36,549,711
21,810,314
35,058,694
26,302,354
24,967,326
18,342,078
33,437,741
28,897,182
# Raw readsa
24,233,756
21,791,604
33,844,963
20,225,194
31,991,715
24,181,347
23,169,302
16,972,473
30,823,546
26,737,835
# Valid readsb
92.2%
92.3%
92.6%
92.7%
91.3%
91.9%
92.8%
92.5%
92.2%
92.5%
(%)c
21,289,707
19,699,548
30,416,117
17,749,598
28,556,375
21,510,417
21,135,267
15,365,296
27,985,037
24,229,865
# Mapped readsd
87.9%
90.4%
89.9%
87.8%
89.3%
89.0%
91.2%
90.5%
90.8%
90.6%
(%)e
19,026,214
17,821,199
27,617,209
16,392,406
26,185,375
20,048,125
19,706,098
14,233,071
25,946,502
21858844
#Uniquely mapped
readsf
78.5%
81.8%
81.6%
81.0%
81.9%
82.9%
85.1%
83.9%
84.2%
81.8%
(%)g
99.2%
99.3%
98.3%
98.0%
97.8%
98.2%
97.2%
96.7%
97.9%
97.5%
Strandnessh
RNA
TopHat
(Trapnell et al., 2009)
rheMac3
calJac3
TopHat
panTro4
mm10
rn5
v2.0.8b
TopHat
(Anders et al., 2015)
HTSeq
2,000 bp
HTSeq
v0.6.0
Ensembl Genes Database
protein_coding
ncRNA
HtH
2
pancRNA-partnered gene
rheMac3
Ensembl Genes Database
UCSC Genome Browser Database
74
LiftOver
rheMac2
Ensembl Genes Database
rheMac3
(Fujita et al., 2010; Meyer et al., 2013)
DEGES
(Sun et al., 2013)
Spearman
1
R
Ensembl Compara
1
(Flicek et al., 2013)
pancRNA-partnered gene
mRNA
pancRNA
(Pearson
pancRNA-lacking gene
pancRNA
(Pearson
pancRNA-partnered gene
0.7
mRNA
0.4
)
pancRNA
mRNA
(Pearson
)
4
(Pearson
)
0.7
0.4
)
Spearman
1
1
75
Tissue-specificity Index (TSI)
(Yanai et al., 2005)
TSI
Tukey-Kramer
(mm10)
PhastCons
(Siepel et al., 2005)
DNA
21
Tukey-Kramer
DNA
LASTZ
LASTZ
DNA
2
LASTZ
UCSC Genome Browser Database
Chain Track
76
Net Track
(Meyer et al., 2013)
panTro4
calJac3
rn5
5
calJac3
rn5
mm10
Mbp
2,000 bp
5
DEGES
Tukey-Kramer
FDR < 0.05
5
14.5
ICR
(Mus musculus; Japan SLC)
4.17 mM
Mg2+
HBSS (SIGMA)
77
NaHCO3
Ca2+
poly-L-ornithine
bFGF
fibronectin
10ng /ml
1% Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque)
N2-supplemented DMEM-F12 medium (Life Technologies)
37
CO2
1
2
3
4
5
0.5% FBS
N2 medium
4
7
17.5
shRNA
1
RNA
1
shRNA
Table 1
RT-PCR
1
Table 2
78
bFGF
PBS
4%
green fluorescent protein
beta III Tubulin
protein
Tesque)
(Tuj-1; 1:500; Sigma)
glial fibrillary acidic
(1:500; Sigma)
IgG
CF647
(1:1000; Aves Labs)
CF488A
(Biotium)
IgG
CF555
(Biotium)
3
79
IgG
(Biotium)
Hoechst33258 (Nacalai
結果
pancRNA
pancRNA
Directional RNA-seq
1
Directional RNA-seq
Directional RNA-seq
(Cortez et al.,
Directional RNA-seq
2014; Lin et al., 2014; Merkin et al., 2012; Peng et al., 2014)(Table 8)
5
76
60
Directional
RNA-seq
(Table 8)
87.3%
88.7%
(Table 8)
ENCODE project
(Djebali et al., 2012)
80
Directional RNA-seq
5
97.4%
(Table 8)
Spearman
mRNA
0.9
(Figure 11A)
(Figure 11A
Spearman
pancRNA
)
(Figure 11B)
2
0.96
Spearman
pancRNA
81
Kidney 2
Kidney 1
Heart 2
Heart 1
Liver 2
Liver 1
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 1
Cerebellum 2
0.00
0.05
0.10
Height
0.15
0.20
Cerebral Cortex 5
Cerebral Cortex 6
Cerebral Cortex 3
Cerebral Cortex 4
Cerebral Cortex 1
Cerebral Cortex 2
Cerebellum 1
Cerebellum 2
Cerebellum 3
Cerebellum 4
Heart 1
Heart 2
Heart 5
Heart 6
Heart 3
Heart 4
Kidney 5
Kidney 4
Kidney 6
Kidney 3
Kidney 1
Kidney 2
Liver 5
Liver 4
Liver 6
Liver 2
Liver 1
Liver 3
0.00
Liver 2
Liver 1
Kidney 2
Kidney 1
Heart 2
Heart 1
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
Cerebellum 1
0.00
Height
82
0.05
0.10
0.10
Height
0.05
0.15
0.15
0.20
0.20
0.25
Marmoset mRNA
Kidney 2
Kidney 1
Kidney 3
Liver 3
Liver 1
Liver 2
Heart 3
0.05
Heart 2
0.00
0.10
Height
0.10
Height
0.05
0.15
0.15
0.20
0.20
0.25
0.25
Chimpanzee mRNA
Heart 1
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
Cerebellum 1
Liver 2
Liver 1
Kidney 2
Kidney 1
Heart 2
Heart 1
Heart 4
Heart 3
Cerebral Cortex 4
Cerebral Cortex 3
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
Cerebellum 1
0.00
A
Macaque mRNA
Mouse mRNA
Rat mRNA
Figure 11.
Spearman
1
(A)
(B) pancRNA
Liver 2
Kidney 2
Heart 2
Heart 1
Cerebellum 2
Kidney 1
0.30
Liver 1
Cerebellum 1
Cerebral Cortex 2
Cerebral Cortex 1
0.25
0.35
Height
0.40
0.45
0.50
Rat pancRNA
Figure 11. continued
83
Liver 2
Liver 1
Kidney 2
Kidney 1
0.35
0.4
0.40
0.45
0.5
Mouse pancRNA
Heart 2
Heart 1
0.30
0.3
Liver 2
Kidney 3
Kidney 2
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
0.20
Heart 2
Heart 1
Heart 4
0.15
0.2
Liver 1
Heart 3
Cerebellum 1
Cerebral Cortex 4
Cerebral Cortex 3
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
Cerebellum 1
Kidney 2
Kidney 1
0.1
0.30
Heart 3
Heart 1
Liver 2
0.4
0.35
Heart 2
Kidney 1
Liver 3
Liver 1
0.25
Height
0.3
Height
0.40
0.5
0.45
Chimpanzee pancRNA
Cerebral Cortex 2
Cerebral Cortex 1
Cerebellum 2
0.25
Height
Liver 6
Liver 4
Liver 5
Liver 2
Liver 1
Liver 3
0.20
Cerebellum 1
Height
0.2
Cerebral Cortex 3
Cerebral Cortex 4
Cerebral Cortex 1
Cerebral Cortex 2
Cerebellum 1
Cerebellum 2
Cerebral Cortex 5
Cerebral Cortex 6
Cerebellum 3
Cerebellum 4
Kidney 5
Kidney 4
Kidney 6
Kidney 3
Kidney 1
Kidney 1
Heart 1
Heart 2
Heart 5
Heart 6
Heart 3
Heart 4
0.1
B
Macaque pancRNA
Marmoset pancRNA
(Figure 12A)
2
pancRNA
pancRNA
(Figure 12B)
pancRNA
RNA
84
A mRNA
0.5
0.7
0.9
Macaque Kidney
Marmoset Kidney
Chimpanzee Kidney
Rat Kidney
Mouse Kidney
Marmoset Liver
Macaque Liver
Chimpanzee Liver
Rat Liver
Mouse Liver
Chimpanzee Heart
Macaque Heart
Marmoset Heart
Mouse Heart
Rat Heart
Chimpanzee Cerebral Cortex
Chimpanzee Cerebellum
Macaque Cerebellum
Macaque Cerebral Cortex
Marmoset Cerebellum
Marmoset Cerebral Cortex
Rat Cerebellum
Rat Cerebral Cortex
Mouse Cerebellum
Mouse Cerebral Cortex
Figure 12.
Spearman
(
)
(
)
(A) mRNA
(B)
RNA
pancRNA
85
(C) 5
B pancRNA
0.2
0.6
1
Chimpanzee Cerebral Cortex
Chimpanzee Cerebellum
Chimpanzee Heart
Chimpanzee Kidney
Chimpanzee Liver
Marmoset Kidney
Marmoset Heart
Marmoset Liver
Marmoset Cerebellum
Marmoset Cerebral Cortex
Macaque Kidney
Macaque Liver
Macaque Heart
Macaque Cerebellum
Macaque Cerebral Cortex
Mouse Heart
Mouse Kidney
Mouse Liver
Mouse Cerebellum
Mouse Cerebral Cortex
Rat Cerebellum
Rat Cerebral Cortex
Rat Heart
Rat Kidney
Rat Liver
C Conserved pancRNA
−1
0
0.5
1
Macaque Kidney
Rat Kidney
Mouse Kidney
Chimpanzee Kidney
Marmoset Kidney
Mouse Heart
Macaque Heart
Chimpanzee Heart
Marmoset Heart
Rat Heart
Macaque Liver
Mouse Liver
Marmoset Liver
Rat Liver
Chimpanzee Liver
Rat Cerebellum
Mouse Cerebellum
Mouse Cerebral Cortex
Rat Cerebral Cortex
Chimpanzee Cerebellum
Macaque Cerebellum
Marmoset Cerebellum
Macaque Cerebral Cortex
Chimpanzee Cerebral Cortex
Marmoset Cerebral Cortex
Figure 12. continued
86
pancRNA
pancRNA
(Uesaka et al., 2014)
mRNA
pancRNA
5
mRNA
pancRNA
pancRNA
(p < 0.05)
92.6%
96.7%
1
pancRNA
mRNA
1
2
3
94.1%
95.2%
pancRNA
96.1%
(Table 9)
RNA
pancRNA
pancRNA
(pancRNA-partnered gene)
13.2
18.9
11.2%
10.4%
9.6
pancRNA
1
pancRNA
pancRNA
Sphk1
(Imamura et
DNA
87
Table 9.
pancRNA-partnered genes
pancRNA-partnered genes
Species
Total genes
a
Any correlation pancRNA-partnered genes
b
c
(%)
d
/Total genes (%)
e
Chimpanzee
15,599
2,229
2,063
92.6%
13.2%
Macaque
12,107
1,441
1,356
94.1%
11.2%
Marmoset
17,228
1,877
1,786
95.2%
10.4%
Mouse
17,783
3,418
3,304
96.7%
18.9%
Rat
19,602
1,959
1,882
96.1%
9.6%
a
b mRNA
pancRNA
Pearson
p<
pancRNA
Pearson
p<
Pearson
p<
0.05
c mRNA
0.05
d mRNA
0.05
r > 0.7
pancRNA
r > 0.7
e
pancRNA-partnered genes
88
al., 2001; 2004)
1
pancRNA
pancRNA
pancRNApartnered gene
TSI (Tissue-specificity Index)
TSI
TSI
TSI
pancRNA-partnered gene
0.05; Figure 13)
TSI
(p <
pancRNA
(pancRNA-lacking gene)
gene
TSI
TSI
(p < 0.05)
pancRNA-partnered
pancRNA-partnered gene
(HtH
)
TSI
pancRNA-partnered gene
TSI
(p < 0.05)
5
pancRNA
TSI
0.9
pancRNA-partnered gene
(Figure 14A, B)
89
pancRNA
Chimpanzee
Macaque
Marmoset
Mouse
Rat
Tissue-specificity index
1.00
0.75
Total genes
Genes containing other genes
within their promoters
pancRNA-partnered
genes
pancRNA-lacking
genes
0.50
0.25
0.00
Figure 13.
Tissue-specificity index
(
)
Total genes
Genes containing other genes within their promoters
(
pancRNA-partnered genes
genes
pancRNA
pancRNA
(
(
)
90
)
)
pancRNA-lacking
(
pancRNA
(
(TSI > 0.9)
(B)
)
Mouse
)
91
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
Mouse
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
Marmoset
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
pancRNA-lacking genes
Marmoset
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
pancRNA-lacking genes
Macaque
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
pancRNA-lacking genes
Macaque
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
pancRNA-lacking genes
Chimpanzee
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
Organ distribution
Chimpanzee
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
B
pancRNA-lacking genes
Total genes
Genes containing other
genes within their promoters
pancRNA-partnered genes
Organ distribution
A
1.00
Rat
0.75
Cerebral Cortex
0.50
Cerebellum
Heart
Kidney
0.25
Liver
0.00
1.00
Rat
0.75
Cerebral Cortex
0.50
Cerebellum
Heart
Kidney
0.25
Liver
0.00
Figure 14.
pancRNA-partnered gene
(A)
Total genes
(
(
)
pancRNA-lacking genes
)
pancRNA
Genes
containing other genes within their promoters
pancRNA-partnered genes
pancRNA
pancRNA
pancRNA
pancRNA
pancRNA
21
PhastCons
pancRNA-partnered gene
pancRNApartnered gene
pancRNA-lacking gene
4
3
(p < 0.001; Figure 15)
pancRNA-partnered gene
pancRNA-lacking gene
(p < 0.05)
pancRNA
92
PhastCons score
1.00
CDS
0.75
Promoter
Promoter
(pancRNA-partnered genes)
Promoter
(pancRNA-lacking genes)
0.50
0.25
0.00
Figure 15.
mm10
CDS
(
)
21
(
PhastCons score
)
Promoter
Promoter (pancRNA-partnered genes)
(
)
pancRNA-partnered genes
Promoter (pancRNA-lacking genes)
(
)
93
pancRNA-lacking genes
pancRNA
pancRNA
pancRNA
pancRNA
pancRNA
pancRNA-partnered gene
8.1%
11.2%
partnered gene
5.0%
10.3%
6.3%
(Table 10)
Table 11
pancRNA-
(
)
pancRNA
DNA
pancRNA
bp
50 bp
DNA
(McLean et al., 2011;
DNA
Sasaki et al., 2008)
DNA
pancRNA-partnered gene
pancRNA-partnered gene
DNA
1.7%
94
Table 10.
Species-specific pancRNA-partnered genes
Species-specific
pancRNA-partnered genes
Species
pancRNA-partnered genes
Species-specific
(One-to-one orthologs) pancRNA-partnered genes
(%)
/ Total genes (%)
a
b
1,447
117
8.1%
0.75%
928
96
10.3%
0.79%
Marmoset
1,238
78
6.3%
0.45%
Mouse
2,251
251
11.2%
1.41%
Rat
1,307
65
5.0%
0.33%
Chimpanzee
Macaque
a pancRNA-partnered genes
c
5
b pancRNA-partnered genes
pancRNA
Pearson
c pancRNA-partnered genes
d Total genes
mRNA
r < 0.4
Species-specific pancRNA-partnered genes
Species-specific pancRNA-partnered genes
95
d
Table 11.
pancRNA-partnered genes
Chimpanzee-specific pancRNA-partnered genes
LOC746089
PLEK2
MYO3B
TXNDC3
OPRM_PANTR
EYA3
BDKRB2
HIBCH
AP1S1
LOC463277
CDKN2C
Q6J9M1_PANTR
CRYGB
CPA1
TXNDC3
FRRS1
TRIM69
LOC459913
GIMAP6
AP1S1
CDC14A
MYO1E
DEFB126
CRYGB
CPA1
NGF_PANTR
ITGA11
SLC24A3
LOC459913
GIMAP6
GJA8
STARD5
CDK5RAP1
DEFB126
DOCK5
LYAM1_PANTR
SLC28A1
LOC469932
SLC24A3
ZMAT4
ELF3
MCTP2
WFD12_PANTR
CDK5RAP1
TRIM55
IL24
LOC453949
LOC737961
LOC469932
ATP6V0D2
CAPN9
NOD2
HLCS
WFD12_PANTR
GEM
GDI2
SLC6A2
PNPLA5
LOC737961
WISP1
SLC39A12
NFAT5
ATG7
HLCS
PTGS1
HKDC1
ATP2C2
DCLK3
PNPLA5
EXOSC2
WAPAL
LOC454401
KCNMB3
ATG7
BMP15
HPSE2
RCVRN
ADAMTS3
DCLK3
TBX22
GPAM
ARL4D
AREG
KCNMB3
POF1B
CLRN3
RNF157
HPSE
ADAMTS3
Q8HZD4_PANTR
LDHC
SMAD4
BANK1
AREG
NECAP1
EHF_PANTR
LMNB2
CCDC109B
HPSE
DAPK1
SLC3A2
CREB3L3
ANXA10
BANK1
DCX
CCDC88B
TNFSF14
TMEM173
CCDC109B
CLPSL2
MOGAT2
CD22
CAMK2A
ANXA10
LOC739514
LOC100608161
SNTG2
SLC36A1
TMEM173
CTXN3
GLB1L3
LOC745055
HAVCR2
CAMK2A
KRT40
PDZRN4
LOC459212
DRD1
SLC36A1
PAQR5
LRRC43
LOC742221
CRISP2
HAVCR2
MMP17
CNGA3
SPACA1
DRD1
SPERT
IL1R1
IL20RA
CRISP2
PCCA
STEAP3
OPRM_PANTR
SPACA1
RHOJ
CLASP1
LOC463277
IL20RA
96
Table 11. continued
Macaque-specific pancRNA-partnered genes
OR52W1
MED1
SPINK7
CHRNA9
PROSC
LOC718873
POU6F2
LOC710021
GPR87
CAV3
EIF3G
LOC709223
F13A1
RTTN
LOC702601
CEL
AQP8
LOC702013
LOC721411
LOC711561
KLRD1_MACMU
GPR50
CHAC2
KLHL30
Q9TTU8_MACMU TNFAIP6
MDM1
TMED4
CRISPLD2
HSD17B13
NDST4
TTC18
Q8MJ12_MACMU
PI15
MYSM1
CD5L
KIRREL
CHST9
LOC718439
PCCB
OCA2
TESK2
FRMD7
C9orf174
CALCB
LOC703939
PGPEP1L
KCNN2
SLC14A2
PADI3
SERPINA12
RPS6KL1
PLAC1L
CHRDL2
IQCF1
ASXL3
HJURP
LOC709312
Q09HA9_MACMU
RNF128
LOC715315
Q4G413_MACMU
ACTA1
SNRNP70
GPBP1
SRI
PPEF1
SDR16C5
BCDIN3D
LOC696940
LOC713422
SPATA24
LOC704166
LY6D
LDB2
INSIG2
SLC25A20
OTOP3
ZNF648
STYK1
MAEL
MUSK
ZDHHC4
ATP2A1
LOC711164
Q9BDC4_MACMU Q6H2Y1_MACMU
CPLX4
BANF2
VGLL2
TYOBP_MACMU
SELE
GON1_MACMU
ST18
LIPK
DPEP3
97
Table 11. continued
Marmoset-specific pancRNA-partnered genes
SLC17A7
CRTAM
DCLK1
RERE
CHRNA6
TSNAX
CST11
TEX35
CWH43
NDUFV1
RABGAP1L
TECTB
SUCNR1
DNAJC5B
TTC1
TMEM196
CHRNB4
C1QTNF2
MLEC
UBE2D1
GABRP
SLC35F6
LRRC16A
CHRNA2
PLEKHF1
PRSS16
PDE4DIP
CEP170B
P2RX1
CPQ
PRSS45
MLANA
IL22RA1
BATF2
MARE
PAK6
NMI
IGFBP5
CD74
INTU
DENND2C
NPY2R
NEK10
IL13
THRSP
TREML2
IL4
ENTPD2
COL10A1
KCNN1
CBR4
CLDN1
SLC25A43
SLC25A35
NCF2
ETV5
LIPA
RASSF3
CDKL5
PRSS27
S100A9
CCL16
NRSN1
PSMD7
MAB21L3
DNMT3B
SYNE3
SERPIND1
DENND1A
JAGN1
LAX1
IL12RB2
HTATIP2
INO80B
CSRP3
MTR
CLCA2
CYP1A2
98
Table 11. continued
Mouse-specific pancRNA-partnered genes
Fgf6
Cobl
Htr4
Rab30
Neu3
Il25
Fbxo28
Gtf2i
Glra1
Rgs9
Ccnt2
Plekhb1
Tab3
Plekhh2
Pirt
Sfmbt2
Dlg3
Krt12
Nucks1
Cd19
Krt25
Mtap7d2
Zfp710
Med9
Slc26a3
Arg2
Spna1
Scnn1b
Metap2
Kcnj1
Ndnf
Armc4
Sync
BC002230
Ccdc19
Dock11
Pnpla8
L3mbtl4
Prokr1
Fabp4
Kpnb1
Gpr132
Myoc
Cul4b
Etl4
Uggt2
Arhgap15
E130309F12Rik
Pdcd2l
Ddx46
Tsc1
Syp
Wnt4
Slc35f2
Armcx4
Cadm2
Rps6ka1
Ckmt2
Itgb6
Rs1
2410004B18Rik
Slc26a9
Rlf
Gpr139
Tyr
Pde8b
Baz2b
Map3k15
Lphn3
Tfap2d
Dscam
Arid3c
Ccdc130
Ppp3cb
Atf2
Asb11
Wdtc1
Ppp1r3a
Rictor
Tnfsf18
Apc
Dhrs2
Api5
Plekha2
Slc16a9
Gpr151
Snx21
Olfr76
Fhl2
Cmbl
Vps39
Tenm3
Sh2d4b
Ccdc63
Taf7
Olfr1428
Gprc5b
Rrm2b
Fabp12
Slc7a2
Fgd3
Zfp536
Plekhm3
Gbx1
Slc1a7
Slc38a4
Car3
Pou4f2
Spats2l
Setx
Ncald
Mllt4
Wnt3a
Lsg1
Itch
Cdyl2
Camp
Nkx2-6
Slc6a7
4933402J07Rik
Steap4
Ptk2
Eif4e
Acsbg1
Txnip
Olfr221
Rgs14
Gm572
Lphn1
Zbtb20
Manba
Impg1
Med30
Aplnr
Epx
Gm1043
Igf2bp1
Kansl2
Zdhhc21
Ltf
Cnot4
Ankrd45
Dlg2
Gprin2
4933402N03Rik
Prpf40b
Dab1
Atp2c1
Tasp1
Syt16
Tnrc6a
Gdf2
St3gal1
Gpr115
Pde4b
Ip6k1
Cntn5
Lingo2
Ctif
Ccdc164
Fcrls
Tff1
Dock7
2810474O19Rik
Atp8b1
Wdr81
Hrh1
Zfp449
Zfyve27
Myom1
Edn2
Dhrs7c
Fam190a
Ifit2
Tanc2
Lrrc31
E2f2
Pou4f3
Trit1
Resp18
Kcne1
Pnlip
Gemin6
Zbtb2
Dnahc1
Napg
Dmbx1
Shf
Exo1
Fut10
Nkain3
Fam186b
Akt3
Csf1r
Hp1bp3
Gtf2a2
Spsb1
Chrm3
Col8a2
4930417G10Rik
Vip
Kat5
Hspg2
Vps8
Plcb4
Adamts6
Stk30
Tmem182
Rev3l
Pik3ap1
Rspo1
Atp6v1h
Ttc12
Zfp518b
Capg
Fam150a
Dcn
Kazald1
Ablim2
Sik3
Bcor
Gpr1
Dnahc9
Ifngr1
Sacm1l
Sparcl1
Dynlrb2
Erc2
Myct1
Chst3
Gm872
Cbx2
Prkg2
Rasd2
Mthfd1l
Gpr156
Las1l
Mypn
Xiap
Prickle2
Zcchc11
Il17rd
Mc4r
Sh3rf2
Tbata
Thoc3
8430419L09Rik
Neil2
Spen
Gap43
1700007G11Rik
99
Table 11. continued
Rat-specific pancRNA-partnered genes
Cd82
Skap2
Slc5a9
Rhobtb1
Shisa2
Cyth1
Dao
Slc6a13
Spata4
Fam101a
Tgm2
Fam222a
Snapin
P2rx7
Ttll13
Rwdd2b
Pgk2
Cldn14
Slc9a4
Adipoq
Gdf3
Lnx1
Jup
Epgn
Lman2
Psen2
Zfp580
Cdx4
Tat
Blmh
Fam171a1
Il1rn
Pla2g2f
Gclc
Plk3
Clvs1
Olr48
Utp11l
Slc30a5
Smpx
Eri3
Acox3
Mrps18a
Pdlim2
Scgb1a1
RGD1564345
Olr1462
Dnajc11
Tctex1d1
Arhgap25
Ophn1
Fgr
Hmcn1
Rin2
Eppin
Cald1
Dab2
Ccno
Scmh1
Esm1
A1cf
Scp2
Myoz1
Alg14
Rgs10
100
2.1%
1.3%
2.8%
pancRNA
pancRNA
DNA
pancSh3rf3
pancRNA-partnered gene
3.1%
pancVwa5b2
(Table 10)
pancRNA
pancRNA
2
RNA-seq
pancRNA
(Hofman, 1982)
pancRNA
(TSI > 0.95)
(r > 0.95) pancRNA
pancRNA
mRNA
pancRNA
29
mRNA
14
101
Directional
pancSh3rf3
pancRNA
pancSh3rf3
pancRNA
(Figure 16A,B)
pancSh3rf3
pancVwa5b2
1
pancVwa5b2
pancVwa5b2
Vwa5b2
(Figure 9, 16C, D)
pancSh3rf3
mRNA
shRNA
pancRNA
pancRNA
mRNA
(Figure 17) pancSh3rf3
mRNA
pancRNA
14.5
pancSh3rf3
pancVwa5b2
14.5
ßIII-tubulin
Gfap
pancSh3rf3
ßIII-tubulin
(Figure 18)
pancSh3rf3
102
A
SH3RF3/Sh3rf3
Chimpanzee Macaque
Marmoset
Mouse
Rat
Normarized Expression Level
3000
Cerebral Cortex
Cerebellum
Heart
Kidney
Liver
2000
1000
0
B
pancSH3RF3/pancSh3rf3
Chimpanzee Macaque
Marmoset
Mouse
Rat
Normarized Expression Level
150
100
Cerebral Cortex
Cerebellum
Heart
Kidney
Liver
50
0
Figure 16.
(A) SH3RF3/Sh3rf3
VWA5B2/Vwa5b2
(B) pancSH3RF3/pancSh3rf3
(D) pancVWA5B2/pancVwa5b2
VWA5B2
(C) (D)
103
(C)
C
VWA5B2/Vwa5b2
Chimpanzee Marmoset
Mouse
Rat
Normarized Expression Level
5000
4000
Cerebral Cortex
Cerebellum
Heart
Kidney
Liver
3000
2000
1000
0
D
pancVWA5B2/pancVwa5b2
Chimpanzee Marmoset
Mouse
Rat
Normarized Expression Level
500
400
Cerebral Cortex
Cerebellum
Heart
Kidney
Liver
300
200
100
0
Figure 16. continued
104
0.8
**
**
0.6
0.4
0.2
pancRNA
0
Relative Expression Level
1.0
Relative Expression Level
1.2
1.2
1.0
0.8
1.2
1.2
1.2
1.0
1.0
1.0
0.8
0.6
0.6
0.4
0.4
0.2
0
mRNA
Vwa5b2 locus
0.8
0.8
0.6
0.6
**0.4
0.4
0.2
0.2
0.2
0
0
**
pancRNA
pancRNA
*
**
0
1.2
1.2
1.0
1.0
Control
pancRNA knockdown
0.8
0.8
**
0.6
0.4
0.2
0.2
pancRNA
**
0.6
0.4
0
mRNA
mRNA
Pacsin1 locus
0
y
RNA
Student
105
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
t
Gapdh
**
0
0.4
0.2
0.2
Sh3rf3
RT-PCR
p < 0.01;
1.2
mRNA
pancSh3rf3
Figure 17.
Relative Expression Level
Sh3rf3
Pacsin1
locuslocus
Relative Expression Level
b2 locus
pancRNA
0
mR
A
B
Figure 18. pancSh3rf3
pancVwa5b2
14.5
pancRNA
7
(A)
GFP (
250 µm
)
ß-III tubulin ( )
(B) GFP
Gfap (
)
ßIII-tubulin
Gfap
3
0.05
p < 0.01
p<
p < 0.01;
Student
106
t
Gfap
pancVwa5b2
pancRNA
ßIII-tubulin
Gfap
(Figure 18)
107
pancVwa5b2
考察
pancRNA
pancRNA
5
5
Directional RNA-seq
pancRNA
(Figure 12A)
(Brawand et al., 2011; Chan et al., 2009; Jordan et al., 2005)
(Figure 12B)
pancRNA
pancRNA
pancRNA
(Figure 11)
pancRNA
mRNA
5
pancRNA
108
mRNA
pancRNA
(Figure 12C)
pancRNA
ncRNA
5
pancRNA
2
pancRNA
DNA
pancRNA
pancRNA
pancRNA-partnered gene
mRNA
mRNA
pancRNA
pancRNA-partnered gene
5
pancRNA-partnered gene
pancRNA-partnered gene
9.6~18.9%
(Table 9)
pancRNA
pancRNA-partnered gene
pancRNA-partnered gene
pancRNA
mRNA
109
pancRNA-partnered gene
5
pancRNA-partnered genes
pancRNA-partnered gene
pancRNA-lacking gene
(Figure 13)
pancRNA
(Imamura et al., 2004; Tomikawa et al., 2011)
Sphk1
pancRNA
(Imamura et al., 2001; 2004)
DNA
pancRNA-partnered gene
Vim
Nefl
Pacsin1
1
(Tomikawa et al., 2011; Uesaka et al., 2014)
pancRNA-
partnered gene
(Figure 14)
pancRNA-partnered gene
pancRNA
pancRNA
110
pancRNA-partnered gene
pancRNA
pancRNA
pancRNA
65~251
mRNA
pancRNA
pancRNA
pancRNA-partnered genes
TSI
4
pancRNA-partnered genes
TSI
(Figure 19)
1
pancRNA
pancRNA
pancSh3rf3
pancRNA
(Figure 16)
pancVwa5b2
14.5
2
pancRNA
(Figure 9, 17) Sh3rf3
pancRNA
40%
shRNA
111
Vwa5b2
Tissue-specificity index
1.00
0.75
Species-specific
pancRNA-partnered genes
Orthologous genes of
species-specific
pancRNA-partnered genes
0.50
0.25
0.00
Figure 19.
pancRNA-partnered gene
5
pancRNA-partnered gene
pancRNA-partnered gene
TSI
(
)
112
4
TSI (
)
5
pancRNA
pancRNA
pancRNA
mRNA
pancRNA
pancRNA
pancSh3rf3
pancVwa5b2
(Figure 18)
pancRNA
pancRNA
1
Sh3rf3
(Wu et al., 2013) (Figure 20)
Vwa5b2
Sh3rf3
Vwa5b2
Sh3rf3
Sh3rf3
Vwa5b2
Sh3rf3
pancRNA
Sh3rf3
Vwa5b2
113
Vwa5b2
Vwa5b2
A
B
Figure 20.
SH3RF3/Sh3rf3
VWA5B2/Vwa5b2
SH3RF3
(A)
Sh3rf3
(B)
(C)
VWA5B2
Vwa5b2
114
(D)
B
Figure 20. continued
115
C
D
Figure 20. continued
116
D
Figure 20. continued
117
Sh3rf3
Sh3rf3
Vwa5b2
Vwa5b2
(Figure 21)
Vwa5b2
Sh3rf3
JNK
JNK
(Hirai et al., 2011; 2002;
Kärkkäinen et al., 2006; 2010; Nix et al., 2011)
Sh3rf3/pancSh3rf3
Vwa5b2/pancVwa5b2
pancRNA
pancRNA
DNA
ncRNA
pancRNA
pancRNA
pancRNA
21
(Figure 15)
pancRNA-partnered gene
pancRNA-lacking gene
< 0.05)
(p
pancRNA
118
Vwa5b2
Sh3rf3
150
Normarized Expression Level
Normarized Expression Level
1500
1000
500
E11 NSC
E14 NSC
E14 NSC (4DIV)
100
E18 NSC
Neuron
Astrocyte
Oligodendrocyte
50
0
0
Sh3rf3
Figure 21.
11.5
(E11 NSC)
(E14 NSC)
(E14 NSC 4DIV)
(Neuron)
4
Vwa5b2
14.5
14.5
18.5
(E18 NSC)
(Astrocyte)
(Oligodendrocyte)
119
pancRNA
(Figure 22)
DNA
RNA
DNA
pancRNA
pancRNA
pancRNA
120
A
mRNA
Promoter region
5'
3'
Sequence
diversity
B
++
−
• Mutaion
• bGCC
• Deamination of
5-methylcytosin
mRNA
Promoter region
5'
3'
Sequence
diversity
C
++
−
mRNA
R loop structure
5'
3'
pancRNA-mediated
transcription regulation
pancRNA
mRNA
5'
3'
pancRNA
Sequence
diversity
+
−
Figure 22. pancRNA
(A) pancRNA
(B) pancRNA
(Mutaion)
GC-biased gene conversion (bGCC)
(Deamination of 5-metylcytosin)
GC
(C)
pancRNA
pancRNA
pancRNA
R
mRNA
mRNA
121
pancRNA
総括
1
pancRNA
lncRNA
pancRNA
pancRNA
pancRNA
CGI (CpG
)
CCG
2
5
pancRNA
pancRNA
pancRNA
pancRNA
(Figure 22)
pancRNA のレパートリーの進化
2
pancRNA
pancRNA
(Figure 22A)
CGI
pancRNA
bp
122
50 bp
pancRNA-partnered gene
DNA
DNA
pancRNA
DNA
pancRNA
NECAP1
pancRNA
NECAP1
5
2
NECAP1
DNA
pancRNA
NECAP1
(Murshid et al., 2006)
NECAP1
(Alazami et al., 2014)
NECAP1
pancRNA
pancRNA
1
pancRNA
CG
GC
pancRNA
123
GC
GC-biased gene conversion
(Galtier and Duret, 2007; Galtier et
al., 2009)
DNA
(Lander et al.,
GC
2001)
CGI
pancRNA
(Figure 22B)
(Khaitovich et al., 2005)
partnered gene
2
pancRNApancRNA
pancRNA
pancRNA
124
pancRNA の作用機序解明が多様性研究に与えるインパクト
pancRNA
pancRNA
1
RNA-DNA
DNA-
R
(Ginno et al., 2012; Uesaka et al.,
2014)
pancRNA
pancRNA
pancRNA
(
1
)
HOTTIP
RNA
pancRNA
RNA
DNA
R
(Figure
22C)
pancRNA
pancRNA
125
pancRNA の比較オミックス解析がもたらす RNA 研究の発展
ncRNA
lncRNA
RNA-seq
pancRNA
lncRNA
RNA
mRNA
isoform
poly A
(Barbosa-Morais et al.,
2012; FANTOM Consortium and the RIKEN PMI and CLST (DGT), 2014; Merkin
et al., 2012; Necsulea et al., 2014; Ozsolak and Milos, 2011)
5’
RNA
3’UTR
RNA
piRNA
(Araya et al., 2014; Brown et al., 2014;
Ho et al., 2014; Yue et al., 2014)
pancRNA
pancRNA
126
ncRNA
結語
pancRNA
127
謝辞
(GAIN)
RNA-seq
128
COE
129
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