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RNA干渉 RNAi (interference)
• 2006年ノーベル生理学医学賞
RNA干渉-二重鎖RNAによる遺伝子サイレンシ
ング-の発見 (1998)
Andrew Fire
Craig Mello
同年ノーベル化学賞：Roger Kornberg
(Arthur [1959生理学医学賞(41歳): DNA合成酵素
の研究] with Severo Ochoa de Albornoz)の長男
真核生物の転写の分子的基礎に関する研究
Gene silencing by 20-22 nt RNA
fragments
線虫では，餌からも
mRNA-like
(RNase III)
mRNA分解，翻訳阻害 ＋ 転写阻害
siRNAs: 21-23 nucleotide double
stranded RNA (dsRNA) duplexes with
symmetric 2-3 nucleotide 3’ overhangs
and 5’ phosphate and 3’ hydroxyl
groups.
転写酵素RNA polymeraseは３種類
I rRNA用
II 普通のタンパク遺伝子mRNA+snRNA
(U6以外), miRNA いくつかのsnoRNA
III 5S rRNA, tRNAその他多数の小RNA
snRNA: mRNAのスプライシング
snoRNA: rRNAの修飾（塩基部分の加工）など
sn: small nuclear, sno: small nucleolar
RNAについて整理しておこう
mRNA processing by snRNP
rRNA processing by snoRNP (cleavage and base
modification)
tRNA processing by a Ribozyme RNase P as a
transcription factor for RNA polymerase III
SRP, TelomeraseのRNA成分
その他の遺伝子発現調節RNA: miRNAs, siRNA,
piRNA, lncRNA: long non-coding RNA≒lincRNA: long
intergenic non-coding RNA, Enhancer/Promoter RNA,
etc.
Small RNAs
U: uridine-rich
Small nuclear RNA (snRNA)
U1, U2, U4, U5, U6: Components of major spliceosome
U4acat, U6acat, U11snRNA, U12 snRNA: Component of minor spliceosome
U7 snRNA: Histone mRNA transcriptional termination
Small nucleolar RNA (snoRNA) About 200 types
U3 snoRNA, U8 snoRNA: rRNA processing
various box C/D snoRNAs Site-specific methylation of the 2’ OH group of rRNA
various box H/ACA snoRNAs Site-specific rRNA modification by formation of
pseudouridine.
Other RNA classes
7SL RNA Component of signal recognition particle for transporting proteins
through ER Translocon (ribosomeと同じく細胞質で働く)
7SK RNA RNA Component of telomerase
RNA component of RNase P, RNA component of RNase MRP: Processing of
tRNA molecules
XIST RNA Regulatory gene imposing X-chromosome inactivation
H19 RNA Imprinted gene, function unclear
SRA RNA encoding a steroid receptor coactivator
Many kinds of ncRNAs
•
•
•
•
III
I
III
tRNA, rRNA (35S, 5S)
U6 III
snRNAs:
Spliceosome
Also mRNA editing!
snoRNAs: rRNA processing (some found in introns)
Telomerase guide RNA, SRP RNA, RNase P
I in nucleolus 仁
RNAs, ……..
• miRNA, piRNA, rasiRNA (micro, piwi-interacting, repeat associated
small interfering)
• siRNA, shRNA (small inhibitory, short hairpin)
• hnRNAs: (mRNA) and many others coming
(Introns might work as ncRNAs and non-coding regions of mRNA have
regulatory roles.)
hnRNAs (heteronuclear RNAs): formerly thought to be the precursors of mRNAs
miRNAs and new ncRNAs
miRNAs (microRNAs): ～22 nt short chains, derived
from double-stranded or short-hairpin RNA
precursors. ～800 species in humans
Leading to inactivation/degradation of mRNA (in the
cytosol) and/or silencing of corresponding genes (in
the nucleus)
～1/3 of genes are controlled by miRNA!
piRNAs (piwi-interacting), rasiRNA (repeat-associated
small interfering) : 24～31 nt
mRNA-like non-coding RNAs
Stays in the nucleus, their function largely unknown!
Imprinting, Differentiation, Development
http://research.imb.uq.edu.au/rnadb/
http://microrna.sanger.ac.uk/ http://www.narna.ncl.ac.uk
mRNAs: exons, introns & UTRs
Green diamonds represent snoRNAs and
orange triangles represent miRNAs
miRNA: microRNA
遺伝子よりPolymerase II で転写
（あるいは別の転写単位の
Exon/Intron）： pri-miRNA (primary
miRNA)の特異的なloop領域が、
Drosha (RNase III) に認識され
て切断：pre-miRNA
これがExportin/GTP-Ranと結合
して核外輸送
細胞質でDicer (RNase III)に分
解されてmiRNA
Argonauteタンパクと結合して
RISC (RNA-induced silencing
complex)形成
mRNA分解、翻訳制御
Micro or small inhibitory
Dicer: RNase III
RISC: RNA-induced
silencing complex
composed of miRNA
(siRNA) and
Argonaute or its
homologs (RDEs)
miRNA/siRNA-induced gene
silencing (RNAi)
Nature 418, 244-251, 2002
RNA-dependent
RNA polymerase
(RdRP)特に植物
で
Higher plants, in addition, have two non-essential RNA
polymerases, Pol IVa and Pol IVb that specialize in
small RNA-mediated gene silencing pathways, working
together with six single-subunit RNA-dependent RNA
polymerases (RDRs).
1.RNA-directed DNA methylation (RdDM) of
endogenous repetitive elements
2.silencing of transgenes
3.regulation of flowering time genes
4.inducible regulation of adjacent gene pairs
5.spreading of mobile [via plasmodesmata/phloem]
silencing signals (especially for their perception) etc.
Although biochemical details concerning Pol IV enzymatic
activities are lacking, genetic evidence suggests several
alternative models for how Pol IV might function.
Pol IVa may
transcribe dsRNA
generated from
bidirectional
transcripts, including
transcripts of natural
antisense gene pairs,
or dsRNAs resulting
from the annealing of
long-distance mobile
RNAs with target
mRNAs (c and d).
Possible modes of Pol IVa function. Pol IVa may transcribe a
specialized DNA template, such as methylated DNA (a) or
single-stranded RNA transcripts derived from methylated
DNA loci (b).
Muscle differentiation
regulators: serum
response factor and MyoD
A day in the
life
筋細胞の増殖を止め，分
化させるmiRNA, miR-1-1
Science 309:1519, 2005
Binding to and halting the
translation of, Hand2 mRNA, which
encodes the transcription factor for
muscle cell proliferation.
RNA regulates Chromatin
& all the
others
Epigenetics の代表例
& Animals
GENES & DEVELOPMENT
19:1635-1655, 2005
Dosage compensation
雄のX×2
雌の片方×
テロメア・セントロメア領域の不活化
Drosophila
ほ乳類
X染色体
IES: internally
eliminated
sequences
古い大核は
壊される
繊毛虫Cilliate
DNA elimination
genome restructuring
10%–15% of the
germline genome
is eliminated
GENES & DEVELOPMENT 19:1635-1655, 2005
Genome Size and Gene Numbers
Genes
Size
(proteins and
RNAs)
3.2 G
3.4 G
26,854
26,560
Predicted
transcription
units
69,185
71,259
Caenorhabditis 100 M
20,099
28,336
Drosophila
132 M
14,651
17,357
Arabidopsis
1.2 G
～38,000 ?
Homo
Mus
http://www.ensembl.org/ http://www.arabidopsis.org/ The Arabidopsis
Information Resource (TAIR)
Far fewer genes than expected. Alternative transcription start sites, Alt splicing,
(Trans-splicing?)
Mattick: Journal of Experimental
Biology 210, 1526-1547 (2007)
バクテリア(黒)，単細胞（グ
レー），両方（薄青），多細
胞basal (青），植物（緑）
4 largest
脊椎動物
線虫，節足動物
原索
ホヤ
Networking of mRNAs,
Introns, and other
ncRNAs is the driving
force of the
Macroevolution
for
Exquisite response to
the environment and
construction of
complex body plan
Mattick: Journal of Experimental Biology 210, 15261547 (2007)
RT adds –CCC ends
Journal of Experimental
Biology 210, 1497-1506
(2007)
cDNA synthesis and further analysis
Only for those with
poly(A) tail
Nucleic Acids Research 2006 34(2):635-646
Transcriptome解析の方法
Poly(A) tailをもつRNAを逆
転写
多数の配列を効率的に決
定
5’: 158万、3’: 72万、
Start/Stop: 38万＋207万
＋完全長cDNA 10万
Transcriptの配列
解析
以下の図は主に、三浦ら 蛋白質核酸酵素51: 2413, 2006より
SAGE
(Serial analysis
of gene
expression)
PNAS 100, 15776, 2003
マウスのトランスクリプトーム解析
• 44,147 Transcription units
• 47% of which code for proteins (20,000)
• the rest (53%) are non-coding (23,000)
Functional Annotation of Mammalian cDNAs
RNA→DNAの対応
Arabidopsis !
http://omicspace.riken.jp/ARTADE/ (理研 Omic Space)
Transcriptional Forest
Desert
Genes
Homo sapiens 3.2 G
26,854
Predicted
transcription
units
69,185
Mus musculus 3.4 G
26,560
71,259
Caenorhabditis 100 M
elegans
Drosophila
132 M
melanogaster
Arabidopsis
1.2 G
thaliana
20,099
28,336
14,651
17,357
Size
(proteins and
RNAs)
～38,000 ?
Estimated number of miRNAs: 20,000 for mouse and 70,000 for Arabidopsis
動物ゲノムでは
mRNA同様にプロセスされる転写産物
• ヒト：転写産物の1/5のみがタンパクをコード
する(Kapranov 2007)。
• FANTOM3(マウス)では、約一万カ所（＝タン
パク質遺伝子以外の場所）から～35,000の
ncRNAを検出
• さらに、mRNA様でない転写産物も多数
（40%）ある(Cheng 2005)。
Far more transcripts than expected
suggested by Homo and Mus data
• Protein coding genes
Intron
30% of the genome
Exons
1.6%
• Non-coding, non-gene part of genome 68%
→ncRNAs (including antisense transcripts)
60-70% of the genome is transcribed. Pervasive
transcription!!
～98% of the transcripts do not encode protein
In bacteria: riboswitches ～100
In case of yeast: ～10% of transcripts is non-coding (PNAS 103: 17846, 2006) or at the
genome basis, >50% derived from non-coding region (BMC Genomics 2005, 6: 93 )
Not mRNA-like!
蛋白質核酸酵素Vol. 51, p.2413 (2006)
Small
cytoplasmic
Journal of Experimental Biology 210, 1497-1506 (2007)
RNAの暗黒大陸
Mammalian
Transcriptome
The box: Genome
Light green circle:
Detected transcripts
Dark green circle:
Transcripts on both
strands
CDS: protein-coding
sequences, UTRs: 5'- and
3'-untranslated sequences
Major portion of light & dark green
parts might be ascribed to Intron
parts.
Human Molecular Genetics 15: R17-R29, 2006
ポストゲノムどころか
ゲノム比較に関してだけでもやることはいっぱい
その他、個性に関連したSNPなどの多型もある
….comparison of two different methods for localizing promoter regions, one
based on reporter constructs containing sequences around putative transcription
initiation sites (Promoters) and the other involving chromatin
immunoprecipitation (ChIP) with an antibody to RNA polymerase (RNAP) and
hybridization to DNA microarrays….
Science 306: 636-640, 2004
Science 309:1527, 2005
Journal of Experimental Biology 210, 1497-1506 (2007)
Primary-miRNA (=transcript)
Pre-miRNA
Exportin-5－Ran
RISC: RNAmiRNA-RISC
induced silencing
duplex
Activation by ATP
complex
single
Dicer, Argonaute, etc
Rana 2007
repeat-associated siRNAs
miRNA
siRNA
animals, plants, etc
S. pombe and plants
(also RNAs added)
rasiRNA
Drosophila
Transposonの不活化
transposons
RISC: RNAinduced
silencing
complex
RNA-dependent RNA
polymerases (RdRP)
Histone H3 lysine 9 (H3-K9) methylation
and the siRNAs with AGO (RITS complex)
act to maintain heterochromatin!
de-adenylation and decapping (P-bodies as the degradation
site in the cytosol?)
Development 134, 1635-1641 (2007)
RNA duplex
formation on
RISC
Conceptual mechanism for gene
silencing
GENES & DEVELOPMENT
19:1635-1655, 2005
Silencingは5’
方向へ広がる
via RdRP
X-chromosome inactivation
Xist RNA (19 kb) initially transcribed on both Xs
(Comp-strand Tsix is usually expressed In undifferentiated female ES cells )
Covers one of the X chromosomes and silences it.
(=Dosage compensation)
H4 hypoacetylated and H3K27
trimethylated
H3K9 hypermethylated and
H4K20 monomethylated as
well as H2AK119 monoubiquitylated
GENES & DEVELOPMENT 21:11-42, 2007
三毛猫は雌だけ
• X染色体上に茶色（優性）か黒色（劣性）かを
決定する遺伝子があり、ヘテロの個体では、
体表のそれぞれの場所でどちらのX染色体
が不活性化されるかで、その部分が茶色に
なるか黒になるかが決まる。
RNA regulates Chromatin
& all the
others
Epigenetics の代表例
& Animals
GENES & DEVELOPMENT
19:1635-1655, 2005
Dosage compensation
雄のX×2
雌の片方×
テロメア・セントロメア領域の不活化
Drosophila
ほ乳類
X染色体
Epigenetic modification
mitotically heritable!
• 主に、「核内でのDNAの高次構造変化による
遺伝子のサイレンシング」と「細胞質内での
mRNAの分解・翻訳抑制」による非可逆的な
遺伝子発現調節。細胞分裂を経ても娘細胞
に伝えられる。
• 生殖細胞(germ cells)でリセットされるので、
子孫には伝わらないのが普通。
• しかし、Germ cell内で起こった変化は、子孫
に伝わるかもしれない。
細胞分化に関するこれまでの考え方と新しい可能性
Transcription-factor-based and chromatin-based models for differentiation.
Journal of Cell
Science 117, 43554363 (2004)