ISMB読み会2016

RNAiFold2T: Constraint programming design of thermo-IRES switches

Transcript

1. *4.#ಡΈձ http://www.slideshare.net/carushi
2. • Թ౓ʹԠͯ͡ೋ࣍ߏ଄͕มԽ͠ɺRNAͷ࣋ͭػೳΛௐઅ͢ΔRNA termometersͷ ഑ྻΛσβΠϯ͢ΔΞϧΰϦζϜΛ։ൃ • ࢬמΓˍ୳ࡧΛ܁Γฦ͢ݫີղʹՃ͑ͯlarge neighbourhood search algorithmʹΑ ΔᩦཉղΛಘΔ • 42℃→30℃Ͱ50%׆ੑ͕མͪΔThermo-IRESεΠονΛ࣮ݧతʹ΋֬ೝ
3. • ࣮ݧ͸εϖΠϯͷάϧʔϓͱڞಉ • IRESͷߏ଄ղੳ • ͪ͜Βͷ࿦จ͸Թ౓ґଘੑͰ͸ͳ͘ɺೋ࣍ߏ଄Λ յ͢ྨࣅ഑ྻΛσβΠϯ • picornavirusͷdomain 5ʹ͓͍ͯϔΞϐϯϧʔϓͱ ϙϦϐϦϛδϯτϥΫτͷ࿐ग़͕IRESͷ׆ੑʹॏ ཁͰ͋Δ͜ͱΛௐ΂ͨ
4. Science, 2016 Jan. • RNA loop structure, m6A, IRES • 10%ͷmRNA͕capඇґଘͷ຋༁Λड͚Δ • 5’UTR and 3’UTRͲͪΒʹ΋ΤϯϦον
5. RNA folding • ഑ྻ→ߏ଄ • ૬ิతͳԘجͷpairingʹΑΓ҆ఆԽ • A-U, G-C, G-U • ΔG of GC/CG stacking is • 3.4kcal/mol - ATP hydrolysis 7kcal/mol • ࠷҆ఆʢMFEʣߏ଄Λ༧ଌ Hairpin Internal MulV A Exterior Bulge …((…))… Example
6. RNA “inverse” folding • ߏ଄ → ഑ྻ • ಛఆͷMFEߏ଄ΛऔΔ഑ྻΛσβΠϯ͢Δ • RNA߹੒ͷ෼໺Ͱॏཁ • λʔήοτɿ਺े͔Β਺ඦnt ఔ౓ͷΞϓλϚʔɺϦϘεΠον • φΠʔϒʹ΍Δͱ͍͍ͩͨܭࢉ͸ܹॏ • ഑ྻΛ࡞Δ → MFEߏ଄༧ଌ O(N^3) → ഑ྻΛ࡞Δ…ͷ܁Γฦ͠ • γϡʔυϊοτҎ্ͷෳࡶͳߏ଄ͷMFE͸NP׬શͳͷͰେମ͸ແࢹ
7. طଘιϑτ΢ΣΞ Թ౓ґଘͰ̎ߏ଄ΛऔΔ • Switch Design (Flamm 2001) - adapVve walk • Frnakenstein (Lyngso 2012) - geneVc algorithm • ͲͪΒ΋֘౰͢ΔதͰ୳ࡧΛ͠ ͳ͕Β࠷ద഑ྻΛҰͭग़ྗ͢Δ ॳΊͯݫີղͷྻڍ͕ՄೳͳΞϧΰϦ ζϜΛ࡞ͬͨ Inverse folding • RNAinverse • RNA-SSD • INFO-RNA • MODENA (made in Japan!) • NUPACK-DESIGN (Caltech) • Inv (China) • RNAifold
8. ΞϧΰϦζϜ֓ཁ • RNAifoldΛϕʔεͷ੍໿෇͖ͷInverse folding • ෳ਺ߏ଄ʹର࣮ͯ͠ߦ • ೋ࣍ߏ଄Λ໦ߏ଄ʹ෼ղ • ϊʔυؒͷoverlapɾinclusion • ؔ࿈͢Δϊʔυ͸ಉ࣌ʹ࠷దԽ • ϊʔυͷ୳ࡧॱংΛ֬ఆ ਺ࣈٯʁ ࣮૷ɿC++&OR-tools (CP search engine) ϧʔϓ಺͸͓ͦΒ͘ϚϧνεϨου
9. 1,2: bp and up 0: bp and bp 4,6: bp and paired i 5,6: bp and paired j 7: closing bp
10. RNAifoldͷઓུ ……((……))…………….((((…))).)…… ໨తͷߏ଄ ᶃಠཱͷϊʔυʹ෼ղ NNGNNNNNNNN ↓ AAGAAAAAACUA ↓ ᶄ഑ྻΛ੍໿ʹैͬͯຒΊΔ ԘجରΛ૊Ή৔ॴ͸Ԙجରͷ ϖΞͰɺ૊·ͳ͍৔ॴ͸೚ҙ ͷԘجͰNΛຒΊͯΈΔ .((……)). ᶅRNAfoldͰ໨తͷߏ଄ͱ ߹͏͜ͱΛ֬ೝ ※গͳ͘ͱ΋͜ͷ෦෼഑ྻ͸໨తͷߏ଄ͱҰக͍ͯ͠Δඞཁ͕͋Δ unpairdͷԘج͕पғʹ௥Ճ͞ΕΔ͜ͱͰҧ͏MFEߏ଄ΛऔΓ͏Δ
11. ……((……))…………….((((…))).)…… ᶆ֤෦෼ߏ଄͔ΒಘΒΕͨ഑ྻΛ૊Έ߹Θͤͯ࠷ऴతʹ໨తͷߏ଄ͱ߹͏͔֬ೝ RNAifoldͷઓུ AAGAAAAAACUA ACGGGAAACCCAGA ……((……))…………….((((…))).)…… + Target MFE ෆҰகͷ৔߹ • ਖ਼ղ෦෼ͷBpΛ֬཰తʹݻఆ͠࠶୳ࡧ (LNS) • શύλʔϯࢼ͢ (CP)
12. Թ౓ґଘͰ໨తߏ଄͕̎ͭ͋Δ৔߹ 30℃ͷԘجର()෦෼ΛຒΊΔ 30℃ͷϧʔϓ.෦෼ΛຒΊΔ 30℃ͷڥք෦෼ΛຒΊΔ ಉ࣌ฒߦͰؔ࿈͢Δ10℃ͷԘ جରΛຒΊΔʂ → Φʔόʔϥοϓ͕͋ΔྖҬͰ͸ಉ࣌ʹ࠷దԽʢྫʣ
13. • ׬੒ͨ͠΋ͷ͔ΒMFEߏ଄Λ νΣοΫ͢Δʢ30℃→10℃ʣ ޻෉ʢHeurisVcʣ • Ԙجର͸҆ఆతͳGC͔ΒೖΕͯ ΈΔ • ยํͰ͸૊·ͳ͍΋ͷ͸ٯʹෆ ҆ఆͳ΋ͷ͔Β • ϧʔϓྖҬ͸AU͔ΒೖΕͯΈΔ • ͨͩ͠ϥϯμϜม਺Ͱॱংʹ͸ ༳Β͗ Թ౓ґଘͰ໨తߏ଄͕̎ͭ͋Δ৔߹
14. ݁Ռɹthermo-IRESͷσβΠϯ 42℃ʢ׆ੑߴ͍ʣ 30℃ʢ׆ੑ௿͍ʣ Experimentally validated! • RNAifold2TͰީิΛڍ͛Δ • ҎԼͷબ୒ج४ͰD1,D2ΛηϨΫτ • (a)(b)ͷߏ଄ΛͱΔʢP > 0.2ʣ • ҙਤ͠ͳ͍ߏ଄ͷ֬཰ͷ̎ഒҎ্ • ௚ۙͷυϝΠϯ4ͷߏ଄Λෆ҆ఆԽͤ͞ ͳ͍ Activity 42℃/30℃ WT D1 D2 NC Start codon Py tract • FMDV IRES domain 5 • IRESͷCapඇґଘͳ຋༁͸42℃ͷߏ ଄ͷํ͕Α͘ಇ͘
15. IRES׆ੑͷ࣮ݧతݕূํ๏ • Luc/CAT • 35S labelled Luciferase polypepVde • chloramphenicol acetyl transferase polypepVde • SDS-PAGE → DensitometerͰൃݱྔͷൺΛऔΔ CAT 5’UTR Luciferase Cap
16. ݁Ռɹ༧ଌ݁Ռൺֱ λ phage thermoregulator RF01804 Relative frequency of the cost function of SD→௿͋͘Δඞཁ͸ͳ͍ʁ Black : RNAifoldͷશղ୳ࡧ Λ௕࣌ؒߦͬͨ΋ͷ
17. Cost function Flamm, et al. RNA. 2001. G: partition function →-RTlnp(Ω1)-RTlnp(Ω2) →ͨͩ݁͠Ռ1Ͱ࢖͍ͬͯͨ બ୒ج४ʹྨࣅ
18. ୹ (N<130nt) 30min Supplementary શମతͳྻڍ਺͸RNAiFold2T ղ͚ͨ૯਺͸͋·ΓมΘΒͣ ݁Ռɹthermoregulatorͷྻڍ
19. ௕ (N>130nt) 60min Supplementary ݁Ռɹthermoregulatorͷྻڍ શମతͳྻڍ਺͸RNAiFold2T ղ͚ͨ૯਺͸SD͕एׯ্ճΔ
20. • 24hr૸Βͤͯɺग़ͤΔ͚ͩީิΛग़͢ • ΦʔμʔͰܻ̎Ҏ্ҧ͏→ྻڍʹڧ͍ʢ഑ྻ͸ࣅͯΔՄೳੑ͕ߴ͍ʣ ୹ 24hr ݁Ռɹthermoregulatorͷྻڍ
21. • SHAPEʢ࣮ݧతߏ଄ղੳʣͷ σʔλΛ଍͢ͱߏ଄༧ଌ΋େ ͖͘มԽ͢Δ • কདྷతʹ͸औΓೖΕ͍ͨ
22. ίϝϯτ • ΞϧΰϦζϜͱͯ͠͸طଘͷ΋ͷʹ৽ͨͳ੍໿Λ௥Ճͨ͠΋ͷ • χον͚ͩͲݫີղ͑Β͍→طଘख๏ͷίετؔ਺͸࣮͸Πέͯͳ͍ͷͰ͸ʁ • ࣮༻తʹ͸༷ʑͳheurisVc͕ඞཁ • MFEʹ͔͠ʢ༗ޮʹʣ࢖͑ͳ͍ɺߏ଄ܭࢉͷ࠶ར༻͸0 • ௕͍΋ͷʹରͯͨ͘͠͞ΜͷީิΛग़ͤΔͱ͜Ζʹ༏Ґੑ • ͋ͱ͸ؤு࣮ͬͯݧͱ͍͏ํ޲ੑ • ੜ෺ֶతʹ໘ന͍τϐοΫʹ࢖͑Δʢͨʣ఺͕ධՁ͞Εͨͱ͍͏ҹ৅