Entamoeba histolytica splicing/mRNP proteome

First, the snRNP component U1A was HA-tagged and the intron of the RabX13 gene was MS2-tagged and amoeba transformats were established with such constructs. Next, amoeba transformants were UV cross-linked and their nucler extracts were immunoprecipitated (IP) with anti-HA agarose or MS2-spharose beads. Precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. MS2-IP helped to discriminate the nuclear roles (chromatin-, co-transcriptional-, splicing-related), of the proteins probed.

首先，将小核核糖核蛋白（snRNP）组分U1A进行HA标签化，并对RabX13基因的内含子实施MS2标签化，随后利用此类构建体建立变形虫转化体。接下来，对变形虫转化体开展紫外线交联处理，采用抗HA琼脂糖珠或MS2琼脂糖磁珠对其核提取物进行免疫沉淀（IP）实验。对所得沉淀产物进行串联质谱（MS/MS）分析。MS2免疫沉淀（MS2-IP）可有效区分所检测蛋白质的各类核功能，涵盖染色质相关、共转录相关以及剪接相关功能。