1 平成23年度組織的な若手研究者等海外派遣プログラム帰国報告書

平成２３年度組織的な若手研究者等海外派遣プログラム
帰国報告書（研究留学）
提出日：平成 24 年 2 月 12 日
フリガナ
氏
ウチカワ
名
エミコ
所属
内川瑛美子
大学院人間文化創成科学研究科
研究院研究員
派遣先名
Institute of Genetics and Molecular and Cellular Biology (IGBMC)
（国名）
ストラスブール大学遺伝学･分子細胞生物学研究所 (フランス )
(日本出発日 )
派遣期間
（日本到着日）
平成 23 年 4 月 1 日
～
平成 24 年 1 月 31 日
①
平成 23 年 4 月 1 日から、平成 24 年 1 月 31 日までの 10 ヶ月間、Institute of Genetics and Molecular and
Cellular Biology
（ IGBMC）の Prof. Dino 氏の研究室に所属し、Dr. Ann e-Catherine Dock- Bre geon
氏及び Prof. Klaholz 氏の指導のもと研究を行った。生活面においても多くの貴重な体験をし
た。
5 月には IGBMC の Integrated structural Biology (構造解析グループ) の合宿にて、私の研究成果につ
いて、発表を行う機会があった。外国において、多くの構造関係の研究者の前で発表を行うことは初めてで
あり、緊張したが、良いプレゼンテーションであったと評価を頂き、研究のさらなる発展に向け、多くのア
ドバイスをもらうことができた。同時に、同世代の友人たちのプレゼンテーション能力の高さに感心した。
これ以降、より分かりやすく、聴衆を惹きつけるプレゼンテーションが行えるよう、様々な研究者のセミナ
ーを研究した。
6 月には、IGBMC 創設者である Prof. Chambon 氏の誕生日に伴い、ノーベル賞受賞者 4 人、それに準ずる
世界的に有名な科学者たちの行うセミナーが本研究所で開催され、非常に魅力的な公演を聴くことができた。
それぞれの研究分野の一線を走る研究者の講演はどれも面白く、将来このようなプレゼンテーションをして
みたいと思わせるものばかりであった。
7 月には X 線結晶構造解析のために、初めてフランスグルノーブルにあるシンクロトロン ESRF で実験を
行った。日本の Photon Factory にあるシンクロトロンで実験を行う経験があったが、ヨーロッパのシンク
ロトロンで実験することは初めてであり、貴重な体験になった。
10 月には IGBMC の第三者委員会による審査会が行われた。各研究室のリーダーがコミッティの前で研究
成果（publish されたもののみ）を報告し、評価を得るというものである。コミッティはフランス国内外の研
究者から構成されている。各研究室の審査は学生やポスドクにも公開される。また、ポスドク、学生の立場
や環境について直接コミッティと話し合う機会も設けられ、透明性のある研究環境が作られていることに感
心した。
9 月以降スーパーバイザーの Dr. Dock-Bregeon 氏がパリに移動し、Prof. Klaholz 氏の研究室に所属し
た。本奨学金での派遣期間の終了も近づき、次のポスドクのポジションを得るため、就職活動を行った。将
来どのように研究を続けていきたいか、そのために今、すべきことは何か。を改めて深く考える機会になっ
た。外国での就職活動は自分を英語で表現する非常に良い経験となった。多くの国のポスドクや学生、先生
方のアドバイスにより、自分を’’伝える’’ために何をしなければならないかを学んだ。至らないところ
ばかりであったが少しずつ改善した。今後も常に自分自身を分かりやすく、魅力的に表現できるよう意識し、
研究活動を行っていきたいと思った。
研究所内では、異なった分野の日本人研究者と意見を交わす機会も多くあり、自身の研究、研究分野を
これまでとは別の角度からも見ることができた。非常に優秀な日本人研究者が多く、刺激を受け研究できた。
研究に関しては、ITP での１年、組織派遣での 10 ヶ月という期間を合わせ、長期間に渡り、研究テーマ
に取り組めたが、成果を形にする直前で派遣期間が終わってしまったことが残念だった。しかし、この 10 ヶ
月間で体験し、吸収したことは留学しなければ得られなかったものであったと確信している。
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②
氏名：内川瑛美子
Institute of Genetics and Molecular and Cellular Biology（IGBMC）was founded by Prof. Pierre Chambon
and is famous for the Transcription research. In France, it is the largest research center. In this
institute, our laboratory belongs to the Department of Integrated Structural Biology. Prof. Dino Moras
is also one of the founders of IGBMC and quite famous as a structural biologist for 30 years. He
established the biggest structural biology department in Strasbourg. Fortunately I could belong and
study in his laboratory. My supervisor was Dr. Anne-Catherine Dock-Bregeon. She is the specialist of
structural studies of RNA. Under her supervision, I studied RNA-protein interaction.
Since our institute carries out quite high level of transcription research, my theme is also related
elongation step of transcription.
Transcription is the process of creating an equivalent RNA copy from a DNA sequence. In eukaryotes,
DNA is assembled into chromatin, which maintains genes in an inactive state restricting access to
transcription machinery and its accessory factors. Chromatin is composed of histones, which form a
structure called a nucleosome. Transcription of DNA is carried out by RNA polymerases. Whereas a single
enzyme is responsible for this job in eubacteria and archaea, eukaryotes have three nuclear RNA
polymerases to share the task of transcribing the nuclear genes.
Three RNA polymerases are necessary for far greater complexity of most eukaryotic genomes. Within
the nucleus, RNAP I is responsible for synthesizing most of the large rRNA, RNAP II synthesizes mRNAs
and most of snRNA, RNAP III synthesizes a variety of structural and kinetic RNAs including tRNA, 7SK,
5S rRNA and U6 snRNA. RNAP II transcription cycle can be divided into several steps. First, RNAPII
is recruited to the promoter of a gene, where it forms a pre-initiation complex with the general
transcription factors. Next, RNAPII then enters possessive transcript elongation stage, which ends
when the gene has been completely transcribed.
At the elongation cycle, the polymerase simply behaves like a machine, quickly reading the gene.
However, over the past decade a revolution in this thinking occurred, culminating in the idea that
transcript elongation is extremely complex and highly regulated and, moreover, that this process
significantly affects both the organization and integrity of the genome.
It has been known that RNAP II pauses soon after promoter escape. This promoter proximal pausing
was first described for the Drosophila heat-shock genes and similar mechanisms have been described
in several genes. This indicates that post-recruitment regulation occurs much more often than was
previously assumed.
Promoter-proximal
pausing
is
mediated
by
the
action
of
pause
factors,
these
include
5,6-dichloro-1-b-D- ribofuranosyl benzimidazole (DRB), sensitivity-inducing factor (DSIF) and
negative elongation factor (NELF). The factors that alleviate promoter-proximal pausing by DSIF and
NELF include TFIIS and the positive transcription- elongation factor-b complex (P-TEFb).
Transcription elongation is positively regulated by the P-TEFb. P-TEFb is a heterodimer consisting
of cyclin-dependent kinase 9 (Cdk9) and one of the cyclins. Cdk9 is the kinase and phosphorylates serine
2 within the carboxyl-terminal domain of the largest subunit of RNAP II.
The ultimate aim of my project is to understand how the activity of the positive transcription
elongation factor (P-TEFb) is regulated by a non coding RNA, 7SK. P-TEFb required for transcription
by RNA polymerase II and post-transcriptional processing of transcripts into competent messenger RNAs.
Deregulation of its activity leads to severe failures in human health, such as cancer (leukemia) and
cardiac hypertrophy. P-TEFb is also hijacked by the HIV machinery during AIDS invasion. Among its
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regulatory partners, the 7SK RNA stands as a model of non-coding regulatory RNA. It orchestrates the
activity of P-TEFb, through its interaction with the HEXIM1 protein, which has been associated with
cardiac hypertrophy and breast cancer. Binding of the 7SK snRNA to HEXIM turns it into a CDK9 inhibitor.
The 7SK RNA forms a core 7SK snRNP (small nuclear ribonucleoprotein) with the La-related protein, LaRP7
and MePCE. Spontaneous mutations of LaRP7 and subsequent destabilization of 7SK RNA are associated
with tumorigenesis.
My work was to investigate the interaction of 7SK RNA and LaRP7 protein. In particular, I studied
how 7SK RNA is stabilized by LaRP7 via structural aspects. In this project, the structural information
is poorly understood. 3D structure of 7SK RNA and LaRP7 are not known. Powerful information for further
understanding of this system would be gained by solving the 3D structure. Because of difficulties,
not so many RNA and protein complex structures are reported. Since RNA is flexible, it is important
to see the 3D structure of RNA when it binds to protein.
Results depend upon obtention of crystals, and it is important to think about crystal packing to
design the best protein and RNA candidates to make the crystal. Further more, to enhance the chances
of crystallization, conditions have to be found especially for protein, as stable as possible (soluble
and monodisperse).
It is estimated that only N-terminal domain of LaRP7 has stable structure. According to this
information, first we decided to make crystal of N-terminal domain of LaRP7. But mass spectrometry
result and SAXS studies show that the N-terminal domain of LaRP7 does not have conformation without
3’end of 7SK RNA. Then crystallization has been done 3’end of RNA and N-terminal domain of LaRP7.
From the previous studies, LaRP7 thought to recognize the 3’UUUU-OH sequence of 7SK. 7SK is 331
nucleotides 110 kD RNA and its secondary structure has 4 hairpins. The hairpin 4 contains U-rich sequence
(HP4U) placed in 3’ end of 7SK. For crystallization, We designed several RNA constructs of this HP4U
region.
After having overcome optimization of the protein and RNA condition using several biochemical
analysis (Thermal shift assay and Dynamic light scattering), the crystals were grew in two weeks in
four conditions. But these crystals were not diffracted well. It is difficult to reproduce and takes
long time (1 month to 2 months). I optimized the reservoir and Cryo protectant condition. Finally I
could get 3.5Å diffraction (Fig. 1). But the crystal was not stable to collect enough angle diffraction.
For making more stable crystals, I designed new RNA for crystallization and studied dehydration process
before freezing crystals. I could not reached to detect the structural details of LaRP7 and 7SK RNA.
Fig.1 Density map of LaRP7 N-terminal domain and RNA
In parallel with this crystallization trial, I investigated the interaction between 7SK RNA and
LaRP7 protein by several biochemical assays (1) Electrophoretic Mobility Shift Assay : EMSA (2)
Isothermal titration calorimetry : ITC
(1) The EMSA assays showed free 3’UUUU-OH sequence of 7SK is necessary for LaRP7 binding. Several
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HP4U region of 7SK RNA constructs were made (Fig. 2)
HP4U
GGHP4U
L4
HP4
Fig 2 Several constructs of HP4U region of 7SK RNA
HP4U and GGHP4U have free U-rich sequence but HP4 and L4 have U-rich sequence but U is occupied
by other sequence. The experiments showed HP4U and GGHP4U bind LaRP7 but HP4 and L4 did not. These
results showed LaRP7 recognize free U-rich sequence.
Further EMSA assays using different RNA constructs showed just before hairpin 4 region should have
double strand for making free U-rich sequence (Fig 3). So far, the shape of just before hairpin 4 region
was unclear but my experiments strongly suggest that this region is occupied by 5’end of 7SK RNA.
5’ end
3’ end
Marz, M., et al. (2009)
Fig 3
One of Secondary structures of 7SK RNA. (The region of black circle is the sequence just before hairpin 4 region)
(2) To investigate the affinity of LaRP7 N terminal region and 7SK RNA, the Kd were measured by
Isothermal titration calorimetry (ITC). This experiment also supports that free U-rich sequence is
important for LaRP7 binding. Furthermore, ITC results suggest that not only U-rich sequence but also
hairpin 4 contributes for stabilization of 7SK RNA and LaRP7 complex. To confirm this fact, further
experiments are required.
Compared with results of ITC measurements of HP4U (3’ end has OH) and HP4U (3’ end has cyclic
phosphate) (Fig. 4), the Kd of HP4U (3’ end has OH) against L N-terminal domain of LaRP7 was much
better than HP4U(3’ end has cyclic phosphate) against N-terminal domain of LaRP7. This result is
interesting for general RNA-protein binding.
A
B
Fig. 4 The 3’ end has OH (A) and cyclic phosphate(B)
During 10 months I investigated RNA-protein interaction details through this project. To complete
this project, I need a little more time. But I could show the 3D structure roughly and suggested several
features of 7SK RNA and LaRP7 protein interaction. Additionally I could acquire many skills.
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