A tRNA modifying enzyme facilitates RNase P activity in Arabidopsis nuclei

RNase P is the essential activity that performs the 5' maturation of tRNA precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to understand their integration with gene expression pathways. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/B localize in the nucleus and find that their double knock out mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome wide tRNAseq approach, we observe that TRM1A/B are responsible for the m2,2G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/B mutants for specific tRNAs, in particular, tRNAs containing a m2,2G modification at position 26 that are strongly down-regulated in TRM1A/B mutants. Altogether, results indicate that the m2,2G adding enzymes TRM1A/B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNAs biogenesis. Overall design: Mim-tRNAseq analysis of Arabidopsis wild-type and TRM1A/B mutants核糖核酸酶P（RNase P）是催化tRNA前体5'端成熟的必需酶活。除了携带核酶（ribozyme）的祖先型核糖核酸酶P外，研究人员在真核生物中发现了仅含蛋白的核糖核酸酶P（PRORP）。在人类线粒体中，PRORP需与两种蛋白伴侣形成复合物方可具备功能。在植物中，尽管PRORP酶可单独发挥催化活性，但我们仍对其相互作用网络展开研究，以解析其与基因表达通路的整合机制。 本研究聚焦拟南芥细胞核核糖核酸酶P的PRORP2，探究其功能相互作用。通过免疫亲和策略（immuno-affinity strategy），我们证实PRORP2可在体内（in vivo）与tRNA甲基转移酶（tRNA methyl transferases）TRM1A和TRM1B形成复合物。除参与核糖核酸酶P相关的生物学过程外，这些酶还可与RNase Z发生相互作用。 我们发现TRM1A/B定位于细胞核，且其双敲除突变（double knock out mutation）会导致严重的宏观表型。结合免疫检测（immuno-detections）、质谱（mass spectrometry）与全转录组tRNA测序（transcriptome wide tRNAseq）技术，我们证实TRM1A/B可在体内（in vivo）对70%的胞质tRNA的第26位核苷酸进行m2,2G26修饰。 我们借助全转录组tRNA测序与RNA印迹杂交（RNA blot hybridizations）技术，发现TRM1A/B突变体中特定tRNA的核糖核酸酶P活性受损，尤其是那些在第26位带有m2,2G修饰的tRNA，这类tRNA在突变体中表达量显著下调。 综上，研究结果表明，负责催化m2,2G修饰的TRM1A/B可在体内（in vivo）与细胞核核糖核酸酶P功能协同，参与胞质tRNA生物合成的早期步骤。 整体实验设计：对拟南芥野生型与TRM1A/B突变体开展Mim-tRNAseq分析。