Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2′,3′-cyclic-PO₄ and 5′-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3′-OH,2′-PO₄ and 5′-PO₄ termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2′-PO₄ terminus in the end-joining reaction, whereby the 2′-PO₄ enhances the rates of RNA 5′-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2′-PO₄ installed by RNA ligase is removed by a dedicated NAD⁺-dependent RNA 2′-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD⁺-dependent RNA 2′-phosphotransferase activity in vitro.

中文翻译：

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病原真菌毛霉目 tRNA 剪接酶 RNA 连接酶和 tRNA 2'-磷酸转移酶的表征

真菌 Trl1 是一种重要的三功能 tRNA 剪接酶，可修复和密封具有 2',3'-环状-PO ₄和 5'-OH 末端的 tRNA 外显子。Trl1 由 C 端环状磷酸二酯酶和中央多核苷酸激酶末端修复结构域组成，可生成N 端 ATP 依赖性连接酶结构域密封所需的3'-OH、2'-PO ₄和 5'-PO ₄末端。Trl1 酶存在于许多人类真菌病原体中，并且是抗真菌药物发现的有希望的靶标，因为与哺乳动物 RtcB 型 tRNA 剪接酶相比，其结构域结构和生化机制是独特的。在这里，我们报告毛霉目物种（被世界卫生组织视为高度优先的人类病原体）精心设计了一种非规范的 tRNA 剪接装置，其中单功能 RNA 连接酶与任何末端修复酶分开编码。我们表明，卷枝毛霉RNA 连接酶 (MciRNL) 取代 Trl1 连接酶结构域，在芽殖酵母体内的 tRNA 剪接中具有活性。重组 MciRNL 的生化和动力学表征强调了其在末端连接反应中对 2'-PO _{4末端的要求，从而 2'-PO 4}提高了 RNA 5'-腺苷酸化（步骤 2）和磷酸二酯合成（步骤3）分别为~125倍和~6200倍。在经典的真菌 tRNA 剪接途径中，由 RNA 连接酶安装的剪接点 2'-PO _{4被专用的 NAD} ⁺依赖性 RNA 2'-磷酸转移酶 Tpt1去除。在这里，我们通过酵母中的遗传互补鉴定并确认了来自三个毛霉目物种的 Tpt1 直向同源物的生物活性。重组M. circinelloides Tpt1 在体外具有很强的 NAD ⁺依赖性 RNA 2'-磷酸转移酶活性。