用于帽特异性光交联实验的合成帽状mRNA

Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments.

作者信息

Kowalska Joanna, Martin Franck, Jemielity Jacek

机构信息

Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, Warsaw, 02-089, Poland.

Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084, Strasbourg CEDEX, France.

出版信息

Methods Mol Biol. 2016;1428:31-43. doi: 10.1007/978-1-4939-3625-0_2.

DOI:10.1007/978-1-4939-3625-0_2

PMID:27236790

Abstract

The 7-methylguanosine triphosphate cap present at the 5' ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5' cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize cap-binding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.

摘要

真核生物mRNA 5'端的7-甲基鸟苷三磷酸帽在mRNA表达和代谢中发挥着多种作用。使用定制的化学工具，如合成帽类似物或携带修饰帽结构的RNA，可以显著推进对帽结合伴侣的鉴定和研究。在这里，我们提供了在5'帽内用能够光激活的核苷（6-硫代鸟苷或7-甲基-6-硫代鸟苷）特异性标记mRNA的方案，这些核苷可用于光交联实验，以鉴定或表征帽结合生物分子。我们还描述了用带帽RNA进行交联实验以绘制组蛋白H4帽结合口袋的方案。