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Действие белков YB-1 и РАВР на трансляцию полиа (-) и полиа (+) мРНК YB-1

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В первых двух разделах литературного обзора будут рассмотрены вопросы функционирования двух мажорных белков цитоплазматических мРНП, принимающих участие в регуляции трансляции мРНК YB-1: YB-1 и РАВР. Поскольку наибольший интерес для данной диссертации представляют сведения об участии этих белков в регуляции трансляции, основное внимание будет уделено именно этим функциям. В третьей части обзора… Читать ещё >

Содержание

  • СПИСОК СОКРАЩЕНИЙ
  • ОБЗОР ЛИТЕРАТУРЫ
  • 1. Структура и функции Y-бокс-связывающего белка YB
    • 1. 1. Структура, доменная организация
    • 1. 2. Особенности взаимодействия YB-1 с другими молекулами
    • 1. 3. Функции YB-1 в ядре
    • 1. 4. Функции YB-1 в цитоплазме
    • 1. 5. Внеклеточные функции YB
    • 1. 6. Регуляция синтеза и активности YB
    • 1. 7. Участие YB-1 в эмбриональном развитии и онкологических заболеваниях
  • 2. Структура и функции поли (А)-связывающего белка РАВРС
    • 2. 1. Структура, доменная организация и взаимодействие с другими молекулами
    • 2. 2. Функции
    • 2. 3. Регуляция синтеза и активности РАВР
  • 3. Участие 3' НТО в трансляции
    • 3. 1. Регуляция с участием белков, взаимодействующих с 3' НТО
    • 3. 2. Регуляция трансляции микрорегуляторными РНК, специфически связывающимися с 3' НТО
  • МАТЕРИАЛЫ И МЕТОДЫ
  • 1. Плазмидные конструкции, использованные в работе
  • 2. Методы работы с ДНК
    • 2. 1. Полимеразная цепная реакция (ПЦР)
    • 2. 2. Трансформация Escherichia coli плазмидной ДНК
    • 2. 3. Выделение плазмидной ДНК из Escherichia col
    • 2. 4. Обработка плазмиды рестриктазой
    • 2. 5. J1 игирование Т4 ДНК лигазой
    • 2. 6. Получение меченого фрагмента кДНК (ДНК-зонда) для Northen-блота
    • 2. 7. Электрофорез ДНК в геле агарозы
  • 3. Методы работы с РНК
    • 3. 1. Синтез РНК in vitro
    • 3. 2. Дефосфорилирование РНК
    • 3. 3. Мечение РНК по 5' концу Т4 полинуклеотид киназой
    • 3. 4. Получение РНК, биотинилированной по 3' концу
    • 3. 5. Получение суммарной РНК из лизата ретикулоцитов кролика
    • 3. 6. Получение суммарной РНК из клеток HeLa
    • 3. 7. Получение суммарной кДНК
    • 3. 8. Получение поли (А)-фрагмента
    • 3. 9. Выделение РНК из полиакриламидного геля
    • 3. 10. Определение стабильности мРНК (Northern-блот)
    • 3. 11. Электрофорез РНК в полиакриламидном нативном геле
    • 3. 12. Электрофорез РНК в полиакриламидном геле в присутствии мочевины
    • 3. 13. Электрофорез РНК в геле агарозы в денатурирующих условиях
  • 4. Методы работы с белками
    • 4. 1. Выделение рекомбинантного РАВР
    • 4. 2. Выделение рекомбинантного YB
    • 4. 3. Выделение рекомбинантных GST и GST-Paip
    • 4. 4. Обеднение ретикулоцитного лизата по РАВР
    • 4. 5. Электрофорез белков в полиакриламидном геле в присутствии додецилсульфата натрия
    • 4. 6. Иммуноблоттинг (Western-блот)
  • 5. РНК-белковые методы
    • 5. 1. Метод задержки в геле (гель-шифт)
    • 5. 2. Связывание на нитроцеллюлозных фильтрах
    • 5. 3. Ковалентные сшивки под действием ультрафиолета (УФ-сшивки)
    • 5. 4. Выделение белков на биотинилированную РНК
    • 5. 5. Бесклеточная система трансляции (БСТ) из ретикулоцитов кролика
  • РЕЗУЛЬТАТЫ
  • 1. Исследование механизмов специфического действия YB-1 и РАВР на трансляцию А (-) мРНК YB
    • 1. 1. Регуляторный элемент необходим для специфической регуляции трансляции мРНК YB-1 под действием YB-1 и РАВР
    • 1. 2. YB-1 ингибирует, а РАВР не влияет на трансляцию мРНК YB-1 с удаленным сайтом связывания РАВР
    • 1. 3. YB-1 и РАВР не влияют на трансляцию мРНК YB-1 с удаленным сайтом связывания YB
    • 1. 4. YB-1 ингибирует, а РАВР не влияет на трансляцию мРНК YB-1 с разделенными сайтами связывания РАВР и YB
  • 2. Исследование механизмов специфического действия YB-1 и РАВР на трансляцию полиаденилированной мРНК YB
    • 2. 1. Полиаденилирование мРНК YB-1 стимулирует ее трансляцию в лизате ретикулоцитов кролика
    • 2. 2. Полиаденилирование мРНК YB-1 не изменяет набора белков, сшивающихся с 3' НТО мРНК YB-1 в лизате ретикулоцитов кролика
    • 2. 3. YB-1 ингибирует трансляцию А (+) так же как и А (-) мРНК YB
    • 2. 4. РАВР не стимулирует трансляцию полиаденилированной мРНК YB-1 в лизате ретикулоцитов кролика
    • 2. 5. В условиях недостатка РАВР трансляция как А (-), так и А50 мРНК YB-1 зависит от количества РАВР
    • 2. 6. Регуляторный элемент в 3' НТО мРНК YB-1 взаимодействует с поли (А)-фрагментом в присутствии белков лизата ретикулоцитов кролика
    • 2. 7. Влияние РАВР на трансляцию полиаденилированной мРНК YB-1 зависит от длины участка РНК между регуляторным элементом и поли (А)-хвостом
  • ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ
  • 1. Роль YB-1 и РАВР в регуляции трансляции неполиаденилированной мРНК YB
  • 2. Возможные механизмы действия YB-1 и РАВР на трансляцию полиаденилированной мРНК YB
  • 3. Регуляция трансляции мРНК YB-1 и ее место в общей регуляции белкового синтеза
  • ВЫВОДЫ

Действие белков YB-1 и РАВР на трансляцию полиа (-) и полиа (+) мРНК YB-1 (реферат, курсовая, диплом, контрольная)

Многофункциональный Y-бокс-связывающий белок 1 (YB-1) — это ДНКи РНК-связывающий белок с эволюционно консервативным доменом холодового шока. YB-1 принимает участие во многих клеточных процессах, включая пролиферацию, дифференцировку и ответ на стрессовые воздействия (Eliseeva et al.,.

2011). Нокаут гена YB-1 приводит к нарушениям эмбрионального развития и пренатальной гибели животных (Lu et al., 2005; Uchiumi et al, 2006). Участие YB-1 в широком спектре внутрии внеклеточных процессов объясняется, прежде всего, его уникальной способностью взаимодействовать как с ДНК и РНК, так и с различными белками (Eliseeva et al., 2011; Скабкин и др., 2004). Связываясь с поврежденной ДНК, YB-1 принимает участие в репарации ДНК (Guay et al., 2008 bPestryakov et al., 2012). Связываясь с определенными нуклеотидными последовательностями в промоторах генов, YB-1 позитивно или негативно регулирует транскрипцию генов, продукты которых участвуют в делении клеток, апоптозе, иммунном ответе, развитии множественной лекарственной устойчивости и др. (Kohno et al., 2003; Eliseeva et al., 2011).

YB-1 взаимодействует с мРНК на всех этапах ее биогенеза и функционирования (Dong et al., 2009; Evdokimova et al., 2006; Skabkin et al., 2004; Svitkin et al., 2009). В ядре он участвует в регуляции альтернативного сплайсинга пре-мРНК (Chansky et al., 2001; Raffetseder et al, 2003). В цитоплазме упаковывает мРНК в мРНП, регулирует ее трансляционную активность, стабильность и взаимодействие с актиновым и тубулиновым цитоскелетами (Chernov et al., 2008 bDavydova et al., 1997; Evdokimova et al., 2001; Ruzanov et al., 1999; Skabkin et al., 2004). Считается, что YB-1 может регулировать функциональную активность мРНК как напрямую, связываясь со специфическими нуклеотидными последовательностями (Giorgini et al., 2001; Skabkina et al., 2005), так и опосредованно. Во втором случае YB-1 за счет своей РНК-шаперонной активности помогает мРНК образовать правильную вторичную структуру, необходимую для узнавания другими регуляторными белками (Skabkin et al., 2001).

Принимая во внимание, что YB-1 участвует в регуляции многих клеточных процессов, следует ожидать, что его количество строго контролируется. Было показано, что регуляция содержания YB-1 к клетке осуществляется как на уровне транскрипции (Shiota et al., 2008; Yokoyama et al., 2003 b) и трансляции (Fukuda et al, 2004; Hsieh et al, 2012; Kato et al, 2010; Skabkina et al, 2003, 2005; Thoreen et al,.

2012), так и за счет изменения стабильности белка (Chibi et al., 2008; Lutz et al, 2006; Sorokin et al, 2005).

Ранее в нашей лаборатории было показано, что специфическая регуляция синтеза YB-1 может происходить под действием двух мажорных белков мРНП: YB-1 и поли (А)-связывающего белка РАВР. YB-1 избирательно ингибирует трансляцию неполиаденилированной (А (-)) мРНК УВ-1 (авторегуляция) при сравнительно низких концентрациях этого белка, оптимальных для трансляции большинства других клеточных мРНК. РАВР, наоборот, стимулирует трансляцию этой мРНК. Как ингибирование под действием УВ-1, так и стимуляция под действием РАВР наблюдаются на стадии инициации трансляции, на этапе присоединения к мРНК УВ-1 438-преинициаторного комплекса или на предыдущем этапе взаимодействия мРНК с факторами инициации трансляции (БкаЫапа et а1, 2003, 2005). С другой стороны, в 3' нетранслируемой области (НТО) мРНК УВ-1 была обнаружена нуклеотидная последовательность с повышенным сродством к УВ-1 и РАВР. Было показано, что сайты связывания двух белков на этой последовательности перекрываются, а белки УВ-1 и РАВР конкурируют за связывание с ней. Кроме того, было обнаружено, что фрагмент мРНК УВ-1 с этой последовательностью ингибирует трансляцию мРНК УВ-1, а также других мРНК на стадии инициации. Эта нуклеотидная последовательность была названа регуляторным элементом.

Тем не менее, механизмы регуляции трансляции мРНК УВ-1 все еще изучены недостаточно. Данная работа посвящена дальнейшему изучению молекулярных механизмов регуляции трансляции мРНК УВ-1. Для этого были поставлены следующие экспериментальные задачи:

1) выяснить, действительно ли регуляторный элемент (1127−1204 нт) в 3' НТО А (-) мРНК УВ-1 строго необходим для регуляции трансляции под действием УВ-1 и РАВР;

2) выяснить, оказывает ли УВ-1 прямое негативное действие на трансляцию А (-) мРНК УВ-1;

3) выяснить, оказывает ли РАВР прямое позитивное действие на трансляцию А (-) мРНК УВ-1.

4) изучить как влияют УВ-1 и РАВР на трансляцию полиаденилированной (А (+)) мРНК УВ-1.

5) проверить, взаимодействует ли регуляторный элемент в 3' НТО мРНК УВ-1 с поли (А)-фрагментом;

6) выяснить, как зависит влияние РАВР на трансляцию А (+) мРНК УВ-1 от длины и нуклеотидной последовательности участка РНК между регуляторным элементом и поли (А)-хвостом (1329−1503нт).

ОБЗОР ЛИТЕРАТУРЫ.

В первых двух разделах литературного обзора будут рассмотрены вопросы функционирования двух мажорных белков цитоплазматических мРНП, принимающих участие в регуляции трансляции мРНК YB-1: YB-1 и РАВР. Поскольку наибольший интерес для данной диссертации представляют сведения об участии этих белков в регуляции трансляции, основное внимание будет уделено именно этим функциям. В третьей части обзора литературы будут обсуждаться механизмы регуляции инициации трансляции, в которых принимает участие 3' нетранслируемая область мРНК.

выводы.

В бесклеточной системе трансляции из ретикулоцитов кролика показано, что.

1. Регуляторный элемент в 3' НТО неполиаденилированной мРНК УВ-1 строго необходим для негативной регуляции трансляции этой мРНК под действием УВ-1 и позитивной под действием РАВР.

2. УВ-1 обладает прямым негативным действием на трансляцию мРНК УВ-1, в то время как позитивный эффект РАВР обусловлен вытеснением УВ-1 с регуляторного элемента.

3. Фрагмент мРНК УВ-1 с регуляторным элементом образует комплекс с поли (А)-фрагментом в присутствии белков лизата ретикулоцитов кролика.

4. Влияние РАВР на трансляцию полиаденилированной мРНК УВ-1 зависит от длины участка РНК между регуляторным элементом и поли (А)-хвостом. Трансляция полиаденилированной мРНК УВ-1 с укороченным спейсером стимулируется РАВР, в то время, как трансляция полноразмерной мРНК или мРНК с заменой удаленного участка на неспецифическую последовательность не стимулируется поли (А)-связывающим белком.

5. На основании полученных данных предложена модель регуляции трансляции полиаденилированной мРНК УВ-1 за счет образования 3' концевой циклической структуры, препятствующей позитивному действию РАВР на трансляцию этой мРНК.

БЛАГОДАРНОСТИ.

В заключение я приношу слова благодарности всем, кто помогал мне в данной работе. В первую очередь, я признательна Льву Павловичу Овчинникову за предоставленную возможность выполнить данную работу в лаборатории регуляции биосинтеза белка, а также за поддержку, помощь, проявленное внимание и дельные советы.

Безмерно благодарна Дмитрию Лябину, моему «микрошефу», за чуткость, доброту, понимание, помощь в овладении множеством экспериментаторских навыков.

Спасибо Вячеславу Адамовичу Колбу за внимание к моей работе, ценные советы и конструктивную критику.

Огромное спасибо Кате Ким, Насте Селютиной, Диме Кретову, Саше Доронину, Даше Мордовкиной, Лире Нигматтулиной, Эльвире Мавлявиевой, Сергею Гурьянову, Максиму Скабкину, Ольге Скабкиной, Диме Полякову, Насте Ишутиновой за практическое и моральное содействие, за готовность всегда прийти на помощь и за создание дружеской атмосферы в лаборатории.

Отдельные слова благодарности хочется произнести в адрес Елены Алексеевны Соболевой и Нины Михайловны Гришиной за техническую помощь, их незаменимый труд и создание уюта в лаборатории.

Отдельное спасибо Евгении Викторовне Серебровой за неоценимую помощь в написании статей, автореферата и диссертационной работы.

А также, огромное спасибо хочется сказать всем моим близким и родным людям: Ване Кулаковскому, Ане Елисеевой, Татьяне Владимировне Кулаковской, Ренате Александровне Кулаковской, Кате Кулаковской, Александр Прокопьевич и Валентине Николаевне Елисеевой за поддержку во время выполнения и написания этой работы.

Благодарю также всех сотрудников Института белка РАН, с которыми я общалась, а также моих друзей и коллег за веру в мою работу и задушевные разговоры.

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