Microsoft PowerPoint - RNA分解系

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1 RNA の分解系 mrnaサーベイランス (Nosense-mediated mrna decay) ナンセンス変異を有する異常なmRNAを見つけて排除する品質監視の仕組み正常な遺伝子変異した異常な遺伝子遺伝子 (DNA) エキソンイントロン異常な位置に生じた終止コドン ( 終止コドンが異常な位置に出現 ) mrna (RNA) 開始コドン終止コドンタンパク質に変換される情報 mrna 上の異常な終止コドンの有無を監視タンパク質に変換される情報が縮小する分解タンパク質機能の発揮正常なタンパク質正常なはたらき異常な小さなタンパク質 ( 多くの場合検出されない ) もしも合成されると病気の原因となる ( 活性が保持されている場合もある ) 1

2 機能喪失型 Loss of function 機能獲得型 Gain of function 遺伝子変異の二つの様式 Recessive 劣性 Dominant active Dominant negative Dominant negative: 多量体複合体として機能するタンパク質の異常は時として正常なタンパク質の働きを押さえる一つの対立遺伝子の変異が正常な対立遺伝子の働きを押さえる優性ナンセンス mrna は寿命が短い酵母 : (Losson and Lacroute, PNAS,1979) Nonsense mutations of the yeast URA 3 gene reduce the messenger level without lowering its instantaneous rate of synthesis. ヒト : (Chang and Kan, 1979) Homozygous beta 0 thalassemia with nonsense mutation. (Nosense-mediated mrna decay) mrna surveillance (mrna の品質監視機構 ) ナンセンスコドンを有する mrna の選択的分解排除機構 (Nosense-mediated mrna decay) mrna surveillance (mrna の品質監視機構 ) ナンセンスコドンを有する mrna の選択的分解排除機構 Wild type allele 遺伝子変異 (nonsense, frame-shift, splicing site) Mutated allele 遺伝子変異転写ミススプライシングミスに対する細胞の防御機構 ( ナンセンス変異を有する異常なmRNAを識別し排除する普遍的な機構 ) 酵母からヒトまで保存転写エラー mrna プロセシングのエラー PTC (premature termination codon) nonsense codon 遺伝子変異に起因する疾患 ( 遺伝性疾患とがん ) の症状に大きな影響を与えていることが予想される PTC-mRNA start stop 生理的に生ずる異常 mrna の排除機構と考えられる (T 細胞 B 細胞での TCR や Ig 遺伝子組み換えに伴う異常 mrna の排除免疫系における役割 ) PTC 2

3 protects Family A from severe anemia, β-thalassemia(with nonsense mutation at the β-globin gene ) Wild Type intron exon 多量体複合体として機能するタンパク質の異常は時として正常なタンパク質の働きを押さえる一つの対立遺伝子の変異が正常な対立遺伝子の働きを押さえる : 優性に作用 Family A Family B Nonsense mutation Nonsense mutation 2 wild type alleles no symptom Mutated allele Wild type allele Mutated allele Wild type allele no PTC mrna no truncated protein hetero no symptom is working PTC mrna truncated protein hetero anemia does not work 正常対立遺伝子正常対立遺伝子正常対立遺伝子変異対立遺伝子優性抑制型変異 dominant negative mutation 正常な機能機能抑制 β- サラセミアではにより mrna が分解されないと重篤な疾患症状を示す PIKKs and the cellular surveillance mechanisms PIKKs: phosphoinositide 3-kinase related kinases ATG exon1 exon2 >50 nt Ter exon3 exon3 genome surveillance ATM ATR Rad3 Rad3 PIKK PIKK 1000aa ナンセンス変異が存在すると不安定ナンセンス変異が存在しても安定 DNA-PKcs mrna surveillance CR1 CR2 CR3 CR4 PIKK CR5 CR6 タンパク質が発現しない短いタンパク質が発現する CeSMG1 hsmg-1 Similarity (%) FRBH PIKK FRBH PIKK ヘテロ体は無症状ヘテロ体で貧血の疾患症状が現れる translational surveillance mtor FRB PIKK 3

(mrna の品質監視機構 ) mrna surveillance Nonsense-mediated mrna decay, mrna のどのような特徴が監視されているのか? 細胞はどのような機構でナンセンス mrna を認識するのか? その生物学的な意義は? 1. Molecular Mechianism 2. Manipulation of 3.

4 (mrna の品質監視機構 ) mrna surveillance Nonsense-mediated mrna decay, mrna のどのような特徴が監視されているのか? 細胞はどのような機構でナンセンス mrna を認識するのか? その生物学的な意義は? 1. Molecular Mechianism 2. Manipulation of 3. Pathological Meaning 4. Physiological Meaning その医学的な意義は? の操作はどのような応用を可能とするか? requires Translation and Splicing The exon junction complex Transcription Splicing QuickTimeý Ç² TIFFÅiLZWÅj êlí ÉvÉçÉOÉâÉÄ Ç Ç±ÇÃÉsÉNÉ`ÉÉÇ¾å ÇÈÇžÇ½Ç ÇÕïKóvÇ-ÇÅB EJC: Splicing mark proteins (Maquat JCS, 2005) 4

5 Surveillance complex Translation 通常の mrna 分解経路 Surveillance complex recognize nonsense mrna in a manner dependent on translation and splicing Steady-state translation Pioneer round of translation Nonsense mrna UPF1 UPF2 UPF3 Surveillance complex No ribosome Translation EJC:exon-exon junction complex Splicing mark Non-stop protein decay (Maquat JCS, 2005) Evolutionary conserved proteins required for S. cerevisiae Upf1p Upf2p Upf3p C. elegans SMG-1 SMG-2 SMG-3 SMG-4 SMG-5 SMG - 6 SMG-7 Human SMG-1 Upf1 hupf2 hupf3 SMG-5 SMG - 6 SMG-7 SMG-1 SMG-3 SMG-4 P SMG-2 SMG-2 SMG-5 SMG-6 SMG-7 P structure PIKK DNA/RNA helicase eif4g homology NES, NIS TPR TPR, PIN TPR Philip Anderson S. cerevisiae Upf1p Upf2p Upf3p hupf1 SMG-1 phosphorylates UPF1 C. elegans SMG-1 SMG-2 SMG-3 SMG-4 SMG-5 SMG-6 SMG-7 Human hsmg-1 P hupf1 P hupf2 hupf3 hsmg-5 hsmg-6 hsmg-7 Helicase structure PIKK DNA/RNA helicase eif4g homology NES, NIS TPR PIN TPR P P P P ATPase SQ rich P P P -S-Q- -S-Q- -S-Q P -S-Q

Translation Surveillance complex Steady-state translation No Pioneer round of translation Rate limiting Phosphhorylation of Upf1 (Maquat JCS, 2005)

During pioneer round of translation, ribosome finds PTC to form SURF. If SURF is followed by EJC, SMG-1 phosphorylates Upf1.

6 Translation Surveillance complex Steady-state translation No Pioneer round of translation Rate limiting Phosphhorylation of Upf1 (Maquat JCS, 2005) 1. Molecular Mechianism How prematue termination codon is recognized as nonsense? During pioneer round of translation, ribosome finds PTC to form SURF. If SURF is followed by EJC, SMG-1 phosphorylates Upf1. Remodeling and dephosphorylation of the Upf1 complex. Degradation of RNA 2. Manipulation of 3. Pathological Meaning 4. Physiological Meaning 1. Molecular Mechianism 2. Manipulation of 3. Pathological Meaning 4. Physiological Meaning 6

7 Strategy for the specific inhibition of 1. Inhibitors of hsmg-1(wortmannin, caffeine) 2. Dominant negative SMG/UPF mutants 3. sirna-mediated depletion of SMG/UPF proteins Inhibition of permits: Enhancement of the stability of nonsense mrna Detection of nonsense mrna Disease gene finding Previous method Protein synthesis inhibitors Specific inhibition of permits us to clarify the physiological and pathological role of and provides novel applications to biology and medicine Emetine Nat Biotechnol May;19(5): A strategy for disease gene identification through nonsense-mediated mrna decay inhibition. Noensie EN, Dietz HC. Emetine and actinomycin D Oncogene Jan 22;23(3): Manipulation of nonsense mediated decay identifies gene mutations in colon cancer Cells with microsatellite instability. Ionov Y, Nowak N, Perucho M, Markowitz S, Cowell JK. hsmg-1 inhibitor can suppress the degradation of nonsense mrna p53 gene ATG Cont. Calu6 196PTC N PTC Wort. CHX N PTC Cont. A549 WT Wort. CHX TER 500 bp Cell lines p53 allele p53 mrna Nat Genet Sep;36(9): Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer. Huusko P, et al. Hela Cells GAPDH mrna p53 tumor suppressor protein 7

Inhibition of permits: hsmg-1 inhibitor can enhance the expression of the truncated protein encoded by the nonsense mrna Enhancement of the stability of nonsense mrna Detection of nonsense mrna

8 Inhibition of permits: hsmg-1 inhibitor can enhance the expression of the truncated protein encoded by the nonsense mrna Enhancement of the stability of nonsense mrna Detection of nonsense mrna Disease gene finding Calu6 196PTC N PTC A549 WT Cell lines p53 allele p53 protein Enhancement of the expression of truncated protein Detection of mutated protein Estimation of the molecular weight of the truncated protein Estimation of the position of mutation (kda) CBB Cont. Wort. CHX Cont. Wort. CHX Cont. Wort. CHX p53 wt p p actin Hela Cells p53 tumor suppressor protein 1. Molecular Mechianism 2. Manipulation of 3. Pathological Meaning 4. Physiological Meaning is primarily a cellular defence mechanism against a dominant negative effects of truncated proteins Inhibition of causes: Change recessive phenotype to dominant one Exploration of the hidden mutation 8

protects cells from the truncated globin chain in most of the b-thalathemia families Family A(No symptom) globin gene mutation Nonsense codon Family B(β-thalathemia) Mutant allele WT allele mutation

9 protects cells from the truncated globin chain in most of the b-thalathemia families Family A(No symptom) globin gene mutation Nonsense codon Family B(β-thalathemia) Mutant allele WT allele mutation Mutant allele Recessive No abnormal globin mrna No abnormal globin protein Some families show severe phenotype caused by the dominant negative effect of the truncated globin chain Escape Dominant Nonsense codon Abnormal globin chain WT allele Dominant negative does not work depending on the position of nonsense-codon mitigates (weakens) dominant phenotype in C. elegans Wild type smg(-)mutant UNC-54 (Myosin HC, muscle) Not specific for UNC-50 Nonsense mutation in Myosin HC, muscle: Recessive Sometimes Dominant depending on the position of the mutation Mice showing defects in might be used for changing the recessive phynotype to dominant one Can be used for saturation mutagenesis Might provide a novel strategy for the generation of disease model is primarily a cellular protective mechanism against a dominant negative truncated protein Inhibition of changes recessive phenotype to dominant one Exploration of the hidden mutation However, can increase the severity of genetic diseases when the truncated protein retain partial activity. Inhibition of might partially rescue the mutant phenotype (Cellular protective mechanism might be sometimes harmful) 9

Ullrich Disease (Ullrich muscular dystrophy ) strengthens the phenotype of Ullrich disease fibroblasts Ullrich Disease fibroblasts Wild type fibroblasts Collagen type VI α 2 chain gene Frame-shift

Cell-ECM attachment Frame-shift α 2 mrna PTC α 1 chain collagen typevi triple helix α 3 chain Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment α 2 mrna α 1 chain α 2 chain

, Ann Neurol, 55(5), 740-744, 2004) Inhibition of partially rescues the phenotype of Ullrich disease fibroblasts Ullrich Disease fibroblasts collagen, type VI, α 2 gene Frame-shift α 2 mrna PTC

10 Ullrich Disease (Ullrich muscular dystrophy ) strengthens the phenotype of Ullrich disease fibroblasts Ullrich Disease fibroblasts Wild type fibroblasts Collagen type VI α 2 chain gene Frame-shift ---> PTC collagen, type VI, α 2 gene #21q22.3 collagen, type VI, α 2 gene #21q22.3 collagen, type VI, α 2 gene #21q22.3 Collagen TypeVI α 2 chain Triple helix domainr498 α 2 mrna α 1 chain α 2 chain collagen typevi triple helix α 3 chain Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment Frame-shift α 2 mrna PTC α 1 chain collagen typevi triple helix α 3 chain Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment α 2 mrna α 1 chain α 2 chain collagen typevi triple helix α 3 chain Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment α1and α3 chains are not expressed at all (Usuki F et al., Ann Neurol, 55(5), , 2004) Inhibition of partially rescues the phenotype of Ullrich disease fibroblasts Ullrich Disease fibroblasts collagen, type VI, α 2 gene Frame-shift α 2 mrna PTC collagen typevi triple helix Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment #21q22.3 α 1 chain α 3 chain hsmg-1 inhibitor(wortmannin, caffein) hsmg-1 sirna collagen, type VI, α 2 gene Frame-shift α 2 mrna PTC collagen typevi triple helix Secretion and Incorporation into ECM Integrin β1α5 Cell-ECM attachment #21q22.3 α 1 chain α 2 chain α 3 chain (Usuki F et al., Ann Neurol, 55(5), , 2004) Inhibition of partially rescues the defects of Ullrich s disease fibroblasts COLa2 (VI)/G6PDH (% of non-silencing) Col α2(vi) mrna Col VI protein Cell-ECM attachment COLVI 50 0 non-silencing hupf1 sirna hsmg-1 sirna Non-silencing hupf1 sirna hsmg-1 sirna Cell-ECM attachment non-silencing * p<0.001 * hupf1 sirna * hsmg-1 sirna 10

11 TCR and Ig gene rearangements frequently generate nonsense codons 1. Molecular Mechianism 2. Manipulation of 3. Pathological Meaning 4. Physiological Meaning mrna quality control mechanism regulation of gene expression? (natural target?) QuickTimeý Ç² TIFFÅiLZWÅj êlí ÉvÉçÉOÉâÉÄ Ç Ç±ÇÃÉsÉNÉ`ÉÉÇ¾å ÇÈÇžÇ½Ç ÇÕïKóvÇ-ÇÅB How nonsense mrnas are generated? Wild type allele exon intron Mutated allele Gene mutation (nonsense, frame-shift, splice sites) stop codon Errors during transcription Errors during splicing nonsense codon nonsense-mrna start codon nonsense codon stop codon Truncated protein 11

12 Very little is known about the role of in human physiology and diseases However, we now have a variety of tools that can specifically suppress, and it is possible to examine the contribution of in a variety of physiological and pathological states の抑制により本来分解排除される mrna を安定化応用 (1) ナンセンス mrna を生ずる未知の変異遺伝子の探索 ( 疾患の責任遺伝子探索の新手法 ) の抑制により本来合成されない異常タンパク質を発現応用 (2) dominant negativeに作用する異常タンパク質を発現する遺伝子変異の場合 (SMG-1 KOマウスを用いた疾患モデルマウス作成の新戦略?) 応用 (3) 異常タンパク質が正常な活性を示し本来排除される必要のないmRNAが排除されている場合 (が有害な作用を及ぼしている遺伝性疾患の克服?) 応用 (4) 遺伝子変異が集積し細胞の防御システムとしてのの必要性が高い細胞の場合 ( がん細胞? 制がん?) 疾患 ( 変異体 ) の責任遺伝子探索の新手法遺伝子変異の 1/4 1/3 はナンセンスコドンを生ずるタイプである ( 遺伝性疾患がん ) mrnaサーベイランス系の抑制本来排除されるmrnaを安定化変異遺伝子に由来するmRNAの増加 transcriptome 解析 (DNAアレイ) による検出変異遺伝子の発見 mrna サーベイランスの操作技術の応用可能性 ( ナンセンス mrna を生ずるタイプの遺伝子変異 ) 個別の診断が可能に (mrna の品質監視機構 ) mrna surveillance Nonsense-mediated mrna decay, mrnaのどのような特徴が監視されているのか? ナンセンスコドンを特定の位置に有するmRNA その生物学的な意義は? 異常タンパク質の持つ毒性から細胞を守る生理的な遺伝子発現制御機構の一端ともなっている? その医学的な意義は? 様々な遺伝子疾患の症状に大きな影響を与えている細胞はどのような機構でナンセンスmRNAを認識するのか? スプライシング翻訳の最初の段階サーベイランス複合体 Upf, SMG 遺伝子群の操作はどのような応用を可能とするか? 12

A b a B AaBb Ab ab 1 1 AB ab A-b a-b 2.Meiosis recombination Meiosis mitosis crossover recombination bivalent DNA 2

A b a B AaBb Ab ab 1 1 AB ab A-b a-b 2.Meiosis recombination Meiosis mitosis crossover recombination bivalent DNA 2 遺伝と遺伝子 2007 年度参考問題リストの解答例 xxax2002@yahoo.co.jp 2007 50 50 1. Mendel s second law of inheritance 2006 2006 A B A B Mendel s second law of inheritance principle of independent segregation P