Kramer Susanne
Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany.
PLoS Pathog. 2017 Jun 19;13(6):e1006456. doi: 10.1371/journal.ppat.1006456. eCollection 2017 Jun.
5'-3' decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5'-3' exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5'-3' degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1) as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5'-3' exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5'-3' decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5' polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA's 5' end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap.
5'-3'衰变是包括锥虫在内的许多真核生物中主要的mRNA衰变途径。去腺苷酸化后，mRNA被脱帽复合体的nudix水解酶DCP2脱帽，最终被5'-3'外切核糖核酸酶降解。独特的是，锥虫缺乏脱帽复合体所有亚基的同源物，而去腺苷酸化和5'-3'降解过程是保守的。在此，我表明这些寄生虫使用一种ApaH样磷酸酶（ALPH1）作为其主要的mRNA脱帽酶。该蛋白最近被鉴定为一种新型锥虫应激颗粒蛋白，并参与mRNA结合。一部分ALPH1仅与锥虫5'-3'外切核糖核酸酶XRNA共定位于细胞后极的一个特殊颗粒，表明这两种酶之间存在联系。通过RNA干扰耗尽ALPH1是致命的，并导致去腺苷酸化但尚未开始5'-3'衰变的总mRNA大量增加。这些数据表明ALPH1在去腺苷酸化下游和mRNA降解上游起作用，这与它在mRNA脱帽中的功能一致。体外实验表明，重组的、N端截短的ALHP1蛋白，而不是催化无活性的突变体，能使带帽的锥虫剪接前导RNA对酵母Xrn1敏感，但前提是包含一种RNA 5'多磷酸酶。这表明ALPH1的脱帽机制与Dcp2的脱帽机制不同，因为它在mRNA的5'端留下不止一个磷酸基团。这是首次报道真核生物ApaH样磷酸酶的功能,这类酶源自细菌，存在于真核生物界的所有系统发育超群中。真核生物ApaH样磷酸酶的底物尚不清楚。然而，相关细菌酶ApaH的底物二腺苷四磷酸与真核生物mRNA帽非常相似。
