微生物iCLIP2:通过促进蛋白质和RNA稳定性,增强RNA-蛋白质相互作用的映射
Nina Kim Stoffel1, Srimeenakshi Sankaranarayanan1, Kira Müntjes1
1Institute of Microbiology, Heinrich Heine University Düsseldorf, Cluster of Excellence on Plant Sciences, 40204 Düsseldorf, Germany.
概括
我们开发了一种用于绘制菌RNA结合蛋白点的新方法. 这项技术确定了与mRNA停止编码子结合的真菌蛋白Rrm4,将RNA运输与线粒体功能联系起来.
科学领域:
- 分子生物学分子生物学
- 菌类学 菌类学是指菌类学.
- 遗传学 是一个遗传学.
背景情况:
- RNA结合蛋白 (RBPs) 调节RNA生命周期,因此需要在体内识别它们的点和结合位点的方法.
- 个人核酸分辨率紫外线交叉链接和免疫沉2 (iCLIP2) 是绘制RBP结合位点的强大技术,但缺乏对微生物系统的优化.
- 由于高的RNase和蛋白酶活性,真菌对iCLIP2提出了独特的挑战.
研究的目的:
- 建立和优化第一个用于真菌的微生物iCLIP2协议.
- 为了确定RBP Rrm4在Ustilago maydis*中的直接RNA标和结合部位.
- 研究Rrm4结合在真菌生物学中的功能影响.
主要方法:
- 对真菌*Ustilago maydis*进行iCLIP2协议的调整和优化.
- 修改机械细胞破坏和溶解缓冲组合,以克服真菌RNase和蛋白酶活动.
- 应用优化的iCLIP2协议来绘制mRNA上的Rrm4结合位点.
主要成果:
- 在Ustilago maydis成功实施微生物iCLIP2方法.
- 交叉链接事件的产量增加,并改善了Rrm4结合地点的识别.
- 发现rrm4特别与线粒体呼吸复合体I,III和V的核编码mRNA的停止编码子结合.
结论:
- 开发的微生物iCLIP2协议作为优化其他具有挑战性的微生物中RBP目标识别的蓝图.
- Rrm4介导的与mRNA停止编码子的结合揭示了内体的mRNA运输和真菌中的线粒体生理学之间的新鲜联系.
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