vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-24-09vavilov-4057Research ArticleГЕНЕТИЧЕСКИЕ РЕСУРСЫ И БИОКОЛЛЕКЦИИPLANT GENETICSHalo-RPD: в поисках мишеней РНК-связывающих белков растенийHalo-RPD: searching for RNA-binding protein targets in plantshttps://orcid.org/0000-0003-3535-3108ШамустакимоваА. О.ShamustakimovaA. O.

Москва

Moscow

nastja_sham@mail.ruВсероссийский научно-исследовательский институт сельскохозяйственной биотехнологииРоссияAll-Russian Research Institute of Agricultural BiotechnologyRussian Federation2024020320242817479Copyright © Шамустакимова А.О., 20242024Шамустакимова А.О.Shamustakimova A.O.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4057

Изучение РНК-белковых взаимодействий и идентификация РНК-мишеней относятся к важнейшим аспектам понимания биологии РНК. К настоящему времени предложены различные методы изучения таких взаимодействий; одним из широко распространенных является иммунопреципитация РНК (RIP). Большинство работ по поиску РНК-мишеней было проведено с использованием антител непосредственно на эндогенный белок или же на GFP, слитый с целевым белком. Зависимость от уровня экспрессии целевого белка и подбор специфичных антител значительно затрудняют классическую иммунопреципитацию. Белок же, слитый с GFP, нередко может быть цитотоксичен, что в дальнейшем приведет к его неправильному узнаванию и/или деградации. В последние годы был разработан ряд мультифункциональных тагов, включая SNAP-tag и HaloTag. Такие таги способствуют изучению целевых белков с разных сторон. Для них созданы флуоресцентные красители, способные прочно связываться с определенным участком тага. Это позволяет как изучать наработку химерного белка, так и определять его локализацию непосредственно в клетке или во всем организме. Для таких тагов разработаны также высокоаффинные субстраты, ковалентно связывающие химерные белки, что значительно сокращает потери в ходе выделения. В данной работе представлен метод, основанный на системе HaloTag, который мы назвали Halo-RPD (HaloTag RNA PullDown). В протоколе используются растения со стабильной экспрессией химерного белка и магнитные шарики Magne® HaloTag® Beads для захвата РНК-белковых комплексов непосредственно из цитоплазматического лизата трансгенных растений Arabidopsis thaliana. Приводится описание основных этапов: 1) подготовка магнитных шариков; 2) гомогенизация тканей и отбор контролей; 3) осаждение и отмывка РНК-белковых комплексов; 4) определение эффективности связывания белка; 5) выделение РНК; 6) анализ полученной РНК. Даны рекомендации для планирования эксперимента по высокопроизводительному секвенированию.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of under-standing RNA biology. Currently, various methods are available to investigate these interactions with, RNA immunoprecipitation (RIP) being the most common. The search for RNA targets has largely been conducted using antibodies to an endogenous protein or to GFP-tag directly. Having to be dependent on the expression level of the target protein and having to spend time selecting highly specific antibodies make immunoprecipitation complicated. Expression of the GFP-fused protein can lead to cytotoxicity and, consequently, to improper recognition or degradation of the chimeric protein. Over the past few years, multifunctional tags have been developed. SNAP-tag and HaloTag allow the target protein to be studied from different perspectives. Labeling of the fusion protein with custom-made fluorescent dyes makes it possible to study protein expression and to localize it in the cell or the whole organism. A high-affinity substrate has been created to allow covalent binding by chimeric proteins, minimizing protein loss during protein isolation. In this paper, a HaloTag-based method, which we called Halo-RPD (HaloTag RNA PullDown), is presented. The proposed protocol uses plants with stable fusion protein expression and Magne® HaloTag® magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic Arabidopsis thaliana plants. The key stages described in the paper are as follows: (1) preparation of the magnetic beads; (2) tissue homogenization and collection of control samples; (3) precipitation and wash of RNA-protein complexes; (4) evaluation of protein binding efficiency; (5) RNA isolation; (6) analysis of the RNA obtained. Recommendations for better NGS assay designs are provided.

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The authors declare that there are no conflicts of interest present.