ジャーナル: Science / 年: 2019 タイトル: A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation. 著者: Sebastian M Fica / Chris Oubridge / Max E Wilkinson / Andrew J Newman / Kiyoshi Nagai / 要旨: During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a ...During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.
ダウンロード / ファイル: emd_4526.map.gz / 形式: CCP4 / 大きさ: 262.9 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Human post-catalytic spliceosome (P complex) stalled with DHX8 S717A mutant, overall map
ボクセルのサイズ
X=Y=Z: 1.2 Å
密度
表面レベル
登録者による: 0.02 / ムービー #1: 0.02
最小 - 最大
-0.08036937 - 0.15654416
平均 (標準偏差)
0.000019515168 (±0.0042213937)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
410
410
410
Spacing
410
410
410
セル
A=B=C: 492.00003 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.2
1.2
1.2
M x/y/z
410
410
410
origin x/y/z
0.000
0.000
0.000
length x/y/z
492.000
492.000
492.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
0
0
0
NX/NY/NZ
208
208
208
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
410
410
410
D min/max/mean
-0.080
0.157
0.000
-
添付データ
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試料の構成要素
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全体 : Human post-catalytic P complex spliceosome
全体
名称: Human post-catalytic P complex spliceosome
要素
複合体: Human post-catalytic P complex spliceosome
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超分子 #1: Human post-catalytic P complex spliceosome
超分子
名称: Human post-catalytic P complex spliceosome / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#44
由来(天然)
生物種: Homo sapiens (ヒト)
分子量
理論値: 3.0 MDa
-
実験情報
-
構造解析
手法
クライオ電子顕微鏡法
解析
単粒子再構成法
試料の集合状態
particle
-
試料調製
濃度
1 mg/mL
緩衝液
pH: 7.9 構成要素:
濃度
名称
式
20.0 mM
HEPES
100.0 mM
KCl
0.2 mM
EDTA
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK III 詳細: 3 uL sample was applied to the grid, left for 25s, then blotted for 2.5-3.5s and immediately plunged into liquid ethane..