Immunoisolation of the trypanosome decapping enzyme ALPH1 delivers a potential subcomplex

Removal of mRNA 5’ caps primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme DCP2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5´-3´exoribonuclease Xrn1. Kinetoplastida lack DCP2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. In our related deposition PXD038550 we analysed the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We showed that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida. The interactome was validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID. Here we carried out affinity capture with T. brucei and T. cruzi ALPH1, which delivers a potential sub-complex missing the C-terminal interactor XRNA.

mRNA 5'帽结构的移除会启动转录本的降解进程，是真核生物基因表达调控的核心环节。经典脱帽酶DCP2（canonical decapping enzyme DCP2）需与5'-3'外切核糖核酸酶Xrn1（5´-3´exoribonuclease Xrn1）组装形成动态多蛋白复合物，进而受到严格调控。动质体目（Kinetoplastida）生物缺乏DCP2的同源基因，而是依赖ApaH样磷酸酶ALPH1（ApaH-like phosphatase ALPH1）完成脱帽过程。该酶由催化结构域与两侧的C端、N端延伸区域共同组成。在我们此前提交的编号为PXD038550的相关数据集研究中，我们通过BioID proximity标记（BioID proximity labelling）技术，对全长ALPH1蛋白及其截短变体开展相互作用组分析，以明确其不同结构域的特异性相互作用。研究证实，布氏锥虫（Trypanosoma brucei）ALPH1可与锥虫XRN1同源蛋白XRNA以及四种动质体目特有蛋白共同构成复合物。我们通过针对布氏锥虫与克氏锥虫（Trypanosoma cruzi）XRNA的亲和捕获反向实验，以及借助BioID技术验证与ALPH1互作的CMGC家族激酶（CMGC-family kinase），完成了该相互作用组的验证工作。本研究通过对布氏锥虫与克氏锥虫ALPH1进行亲和捕获，获得了缺失C端相互作用蛋白XRNA的潜在亚复合物。