1、Protein-RNA InteractionsFunctional roles of RNA binding proteins mRNA localization mRNA translation mRNA splicing mRNA degradation mRNA editing Methods to study protein-RNA interaction EMSA(Electrophoretic mobility shift assays)蛋白质与RNA结合后使其分子量增加 RNA pull-down 下拉RNA，可同时获得其结合的蛋白质 RIP(RNA immunoprecipi
2、tation)免疫沉淀蛋白质，可同时获得其结合RNA CLIP-seq(Cross-Linking and ImmunoPrecipitation)免疫沉淀蛋白质，测序获得其结合位点序列EMSA AUF1 binds to FST 3UTR1,表达纯化蛋白2,蛋白和RNA结合3,电泳RNA pull-downAUF1 binds to FST 3UTR through 112-221nt region1,PCR获得biotin标记的RNA片段1 4411 221222 4411 441codingbiotin2,biotin标记的RNA片段与AUF1结合3,Streptavidin珠子下拉RN
3、A片段4,Western Blot检测下拉产物中是否含有AUF1RIPRIP indenfies AUF1 binding with FST P21 and CyclinD1CLIP-seqTo study the binding sites of a specific proteinIntegrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Processing and mRNA StabilityThe influence of HuR upon target tran
4、scripts begins in the nucleus,where HuR associates with the pre-mRNA introns and affects splicing and likely other nuclear processing events.After assisting with export of the mature mRNA out of the nucleus,HuRs impact continues in the cytoplasm,where it stabilizes mRNAs,may help with transient mRNA storage,and modulates the recruitment of the mRNA with the translation machinery(polysomes).
