РНК-интерференция в терапии рака. Часть 5.

22.04.201120200
МиРНК и микроРНК

Хотя миРНК и микроРНК были открыты независимо друг от друга, оба этих типа малых РНК схожи по биогенезу, сборке в комплексы с белками и способности ингибировать экспрессию генов у различных эукариот (Bartel, 2004; Meister and Tuschl, 2004; Baulcombe, 2004; Mello and Conte, 2004). Dicer, фермент семейства РНКаз III, участвует в генерации обеих этих молекул, разрезая длинную дцРНК, формируя миРНК и разрезая молекулу-предшественницу микроРНК со структурой в виде шпильки, формируя микроРНК. На стадии формирования миРНК и микроРНК имеют вид двуцепочечных дуплексов, которые должны быть расщеплены перед связыванием с комплексом белков RISC.

Оценивая термодинамическую стабильность, миРНК можно разделить на 2 класса: симметричные миРНК и асимметричные миРНК. Концы симметричной миРНК одинаково стабильны, следовательно, обе цепи миРНК с одинаковой эффективностью связываются с комплексом белков RISC (Schwarz et al., 2003). У асимметричной миРНК один конец более стабилен, чем другой. Так как двуцепочечный предшественник миРНК проще расщепить с менее стабильного конца, одна из цепей получает преимущество при связывании с комплексом белков RISC (Schwarz et al., 2003; Khvorova et al., 2003). Интересен тот факт, что большинство миРНК являются высоко асимметричными, что повышает эффективность включения этих молекул в белковые комплексы RISC.

Биохимические исследования in vitro и in vivo показали, что RISC-комплекс с миРНК может функционировать так же, как RISC-комплекс с микроРНК, подавляя трансляцию мРНК-мишени. Аналогично, RISC-комплекс с микроРНК может функционировать так же, как RISC-комплекс с миРНК, расщепляя специфичную молекулу мРНК. Такая функциональная взаимозаменяемость между миРНК и микроРНК говорит о высокой схожести, если не о полной идентичности этих молекул (Hutvagner and Zamore, 2002; Tang.et al., 2003; Doench et al., 2003; Zeng et al., 2003). В то же время, существует множество доказательств, говорящих в пользу того, что миРНК-RISC и микроРНК-RISC представляют собой совершенно разные типы комплексов. Во-первых, биогенез, созревание и последующая сборка миРНК и микроРНК в комплексы проходит совершенно по-разному (Bartel, 2004).Во-вторых, белок Argonaute, неотъемлемый компонент белкового комплекса RISC, кодируется несколькими генами одного семейства и может экспрессироваться в виде различных белковых подгрупп (Carmell et al., 2002; Hammond et al., 2001; Liu et al., 2004; Song et al., 2004; Meister et al., 2004). Различные белки Ago могут придавать соответствующим RISC-комплексам разные свойства и функции. В-третьих, считается, что комплементарность между малыми РНК и их мРНК-мишенями влияет на функциональность RISC-комплекса с точки зрения регуляции стабильности мРНК и трансляции. Тем не менее, RISC-комплексы, содержащие малые РНК, высоко комплементарные к специфичной мРНК-мишени, не всегда осуществляют эффективное разрушение своей молекулярной мишени (Meister et al., 2004). В четвертых, миРНК-RISC-комплексы и микроРНК-RISC-комплексы направлены на разные по функциям мишени клетки. Большинство микроРНК и их RISC-комплексов регулируют экспрессию генов, связанных с ростом и развитием организма (Rhoades et al., 2002). А миРНК получают из дцРНК, синтезируемых in vitro или in vivo из вирусов или генетических последовательностей, получаемых методами генной инженерии. Двуцепочечные РНК также можно получать из эндогенных активированных транспозонов. Таким образом, к биологическим функциям миРНК можно отнести: (1) антивирусную защиту (Pfeffer et al., 2004; Ding et al., 2004), (2) расщепление избыточного количества мРНК в клетке, (3) защиту генома от транспозонов (Mello and Conte, 2004; Hannon, 2002; Tabara et al., 1999).

Заключение

Методы молекулярной биологии, позволяющие искусственно изменять функции генов, дают нам надежду на осознание и понимание регуляторных механизмов, контролирующих функционирование клеток.РНК-интерференция позволяет анализировать функции генов на клеточном уровне и представляет собой эффективную систему.

Источник:
Shafi G., Jamil K., Kapley A.et al.RNAi as a novel therapeutic platform technology for oncological solutions.Biotechnology and Molecular Biology Review 2008; 4(4): 55-70, с дополнениями и изменениями.

перевод: Мария Зайцева, Анаит Григорян

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