Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.
Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.
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DOI：
10.1016/j.xpro.2021.100929
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发表时间：
2021-12-17
期刊：
影响因子：
--
通讯作者：
Gerber AP
中科院分区：
文献类型：
--
作者：
Matia-González AM;Jabre I;Gerber AP
RNA-蛋白质相互作用的体内表征是理解RNA调控机制的关键。在此，我们描述了一个协议，用于检测蛋白质相互作用的多聚腺苷酸化的RNA在酵母酿酒酵母。通过紫外线（UV）照射细胞使蛋白质在体内与核酸交联，并且使用oligo[dT]25珠从细胞裂解物中分离具有结合蛋白质的含poly（A）的RNA。RBP可以通过免疫印迹分析或质谱法检测，以确定mRNA结合蛋白质组（mRBPome）及其在应激下的变化。有关本方案使用和执行的完整详细信息，请参阅。在体内检测与多聚腺苷酸化RNA相互作用的蛋白质紫外线照射细胞以交联RNA-蛋白质相互作用用来自酵母细胞裂解物的oligo[dT]珠捕获多聚腺苷酸化RNA适用于监测mRNA结合蛋白质组的动态变化RNA-蛋白质相互作用的体内表征是理解RNA调控机制的关键。在此，我们描述了一个协议，用于检测蛋白质相互作用的多聚腺苷酸化的RNA在酵母酿酒酵母。通过紫外线（UV）照射细胞使蛋白质在体内与核酸交联，并且使用oligo[dT]25珠从细胞裂解物中分离具有结合蛋白质的含poly（A）的RNA。RBP可以通过免疫印迹分析或质谱法检测，以确定mRNA结合蛋白质组（mRBPome）及其在应激下的变化。
In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to. Detection of proteins that interact with polyadenylated RNAs in vivo UV irradiation of cells to crosslink RNA-protein interactions Polyadenylated RNAs are captured with oligo[dT] beads from yeast cell lysate Suitable for monitoring dynamic changes of the mRNA-binding proteome In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress.
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