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Identyfikacja Domen WG/GW Zaangażowanych W Wiązanie Białek Argonaute Oraz Analiza Mechanizmów Molekularnych Odpowiedzialnych Za Ich Zmienność

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Identyfikacja Domen WG/GW Zaangażowanych W Wiązanie Białek Argonaute Oraz Analiza Mechanizmów Molekularnych Odpowiedzialnych Za Ich Zmienność Wydział Biologii Uniwersytet im. Adama Mickiewicza w Poznaniu Rozprawa doktorska Identyfikacja domen WG/GW zaangażowanych w wiązanie białek Argonaute oraz analiza mechanizmów molekularnych odpowiedzialnych za ich zmienność Andrzej Zieleziński Praca napisana pod kierunkiem Prof. dr. hab. Wojciecha Karłowskiego w Pracowni Bioinformatyki Instytutu Biologii Molekularnej i Biotechnologii Poznań, 2014 Podziękowania Składam serdeczne podziękowania mojemu Promotorowi Panu prof. dr. hab. Wojciechowi Karłowskiemu za intelektualne inspiracje oraz wiedzę, jaką przekazał mi w okresie studiów doktoranckich, a także twórcze rozwinięcie moich zainteresowań naukowych, tak abym mógł przygotować niniejszą dysertację Dziękuję również Koleżankom i Kolegom z Pracowni Bioinformatyki, w szczególności dr. Maciejowi Szymańskiemu, dr. Markowi Żywickiemu oraz mgr Sylwii Alabie za pomoc w kwestiach naukowych, a także pouczające dyskusje Finansowanie Niniejsza praca powstała przy finansowym udziale: 1. Narodowego Centrum Nauki (grant 2011/03/N/NZ2/01440 dla A.Z.) 2. Wydziału Biologii Uniwersytetu im. Adama Mickiewicza w Poznaniu (grant GDWB-09/2011, dla A.Z.) 3. Wojewódzkiego Urzędu Pracy (stypendium w ramach projektu „Wsparcie stypendialne dla doktorantów na kierunkach uznanych za strategiczne z punktu widzenia rozwoju Wielkopolski” w ramach programu 8.2.2. Programu Operacyjnego Kapitał Ludzki) Publikacje autora związane z pracą doktorską 1. Zielezinski A & Karlowski WM. Tyrosine-tryptophan substitution switches on the rapid evolution of WG/GW domain in RRM AGO-binding proteins from Arabidopsis and rice. [publikacja w przygotowaniu] 2. Zielezinski A & Karlowski WM. Whub: a comprehensive knowledgebase portal for AGO-binding protein research Nucleic Acids Research [artykuł wysłany do redakcji] 3. Zielezinski A & Karlowski WM. Agos - -a universal web tool for GW Argonaute-binding domain prediction. Bioinformatics. 27(9). 2011 4. Karlowski WM, Zielezinski A, Carrère J, Pontier D, Lagrange T, Cooke R. Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis. Nucleic Acids Research. 38(13). 2010 Spis treści Streszczenie.......................................................................................................................................6 1. Wstęp.............................................................................................................................................8 1.1. Białka WG/GW w procesie RNAi.........................................................................................9 1.2. Rola domeny WG/GW w wiązaniu białek z rodziny Argonaute.........................................12 1.3. Specyfika domen WG/GW..................................................................................................16 2. Cel pracy.....................................................................................................................................19 3. Metody........................................................................................................................................20 3.1. Metody identyfikacji domen WG/GW.................................................................................20 3.1.1. Metoda kompozycyjna I i II generacji.........................................................................20 3.1.2. Metoda profilu PSSM...................................................................................................22 3.1.3. Metoda wykorzystująca nauczanie maszynowe...........................................................24 3.2. Analiza filogenetyczna.........................................................................................................25 3.3. Technologie wykorzystane w aplikacjach internetowych....................................................27 4. Wyniki.........................................................................................................................................28 4.1. Identyfikacja de novo domen wiążących białka AGO.........................................................28 4.1.1. Metoda przewidywania domen WG/GW oparta na kompozycji aminokwasów.........28 4.1.2. Nowe białka wiążące AGO w genomie Arabidopsis thaliana.....................................33 4.1.3. Wirtualna symulacja eksperymentu wymiany domen WG/GW..................................38 4.1.4. Metoda detekcji pojedynczych motywów wiążących AGO.........................................40 4.1.5. Nowe białka wiążące AGO u Eukariota.......................................................................44 4.1.6. Meta-genomowe przewidywanie domen WG/GW u Prokariota i wirusów.................48 4.2. Programy do adnotacji i analizy domen WG/GW...............................................................56 4.2.1. Whub - portal internetowy do badań nad motywami zaangażowanymi w RNAi........57 4.2.2. Agos - skaner on-line identyfikacji potencjalnych miejsc wiązania AGO...................62 4.2.3. Wsearch / i-Wsearch - programy identyfikacji funkcjonalnych W-motywów.............65 4.2.4. Projektowanie in silico sekwencji domen w formie gry internetowej.........................69 4.3. Molekularne mechanizmy powstawania i zmienności domen WG/GW.............................71 4.3.1. Tandemowe i segmentowe duplikacje genów oraz alternatywny splicing...................73 4.3.2. Tempo mutacji niesynonimicznych i synonimicznych.................................................79 4.3.3. Analiza konwersji genów i/lub rekombinacji...............................................................85 4.3.4. Powstawanie de novo domeny WG/GW......................................................................87 5. Dyskusja......................................................................................................................................91 6. Podsumowanie..........................................................................................................................101 Wykaz skrótów..............................................................................................................................102 Spis rysunków i tabel.....................................................................................................................103 Bibliografia....................................................................................................................................105 Streszczenie Wstęp. Domeny białkowe WG/GW złożone z licznie występujących par tryptofanu (W) i glicyny (G) są niezbędne do wiązania białek Argonaute (AGO) w procesie interferencji RNA (RNAi). Bardzo niski stopień podobieństwa sekwencji domen WG/GW, różna długość ich sekwencji (22-700 reszt), zmienna liczba powtórzeń motywu WG/GW (1-45) uniemożliwiają wiarygodne określenie ich relacji homologicznych, a także są źródłem trudności podczas ich identyfikacji tradycyjnymi metodami przewidywania domen i motywów białkowych. Cel pracy. Celem niniejszej pracy jest identyfikacja nowych białek zawierających domenę WG/GW wiążącą AGO oraz zbadanie mechanizmów molekularnych warunkujących ich zróżnicowanie. Metody. Stworzone programy adnotacji domen wiążących AGO zostały napisane w języku Python. Zakres badań filogenetycznych sprowadzono do konserwatywnych fragmentów białek oddziałujących z AGO. Wyniki. Opracowano trzy metody identyfikacji de novo domen WG/GW (Agos, Wsearch, i-Wsearch) zaimplementowane w formie ogólnodostępnych aplikacji internetowych i programów przeznaczonych do uruchomienia na lokalnym komputerze, które stanowią pierwsze bioinformatyczne narzędzia służące do adnotacji domen wiążących AGO. Wynikiem ich zastosowania są listy rankingowe nowych genów kodujących potencjalne domeny WG/GW u Eukariota, z których część została już potwierdzona eksperymentalnie (WGRP1, SDE3, hnRNP). Skanowanie genomów Prokariota pozwoliło także zidentyfikować sekwencje pierwszych potencjalnych domen WG/GW w tym królestwie, które w większości występują u gatunków archeonów i bakterii kodujących białka Argonaute. Również wśród wirusów - 6 - infekujących rośliny i zwierzęta znalezione zostały statystycznie istotne motywy WG/GW, które mogą oddziaływać z białkami AGO komórki gospodarza w celu przełamania jego systemu odporności zależnego od RNAi. Z rekonstrukcji filogenetycznej i analizy porównawczej domen WG/GW, przeprowadzonej w wielogenowej rodzinie białek hnRNP, wynika, że sekwencje genomowe domen wiążących AGO stanowią ekstremalny przypadek polimorfizmu genetycznego. Oprócz tandemowych i segmentowych duplikacji oraz alternatywnego splicingu, fragmenty sekwencji kodujących motywy WG/GW znajdują się pod działaniem pozytywnej selekcji przyspieszającej utrwalanie substytucji aminokwasowych, które dodatkowo podlegają licznym przetasowaniom między paralogami na drodze częstych rekombinacji i nierównego crossing-over. Przeprowadzając wirtualną symulację eksperymentu polegającego na wymianie domen WG/GW między niespokrewnionymi roślinnymi i zwierzęcymi białkami wiążącymi AGO wykazano, że mimo wysokiego zróżnicowania długości i stopnia zachowania sekwencji tych domen, ich specyficzna kompozycja aminokwasowa jest zachowana u wszystkich organizmów eukariotycznych. Filogenia domen WG/GW oraz analiza ich składu aminokwasowego sugerują, że zarówno zwierzęce, jak i roślinne domeny wiążące AGO, występujące w różnych rodzinach białkowych, powstają z regionów białek inherentnie nieuporządkowanych (IDP, ang. intrinsically disordered proteins) obejmujących także regiony glicynobogate. Ponadto wykazano, że uniwersalną cechą eukariotycznych domen wiążących AGO są wielokrotne - najczęściej
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