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. Mim-tRNAseq analysis of Arabidopsis wild-type and TRM1A/B mutants

核糖核酸酶P（RNase P）是介导tRNA前体5’端成熟过程的必需酶活复合物。除携带核酶的经典型核糖核酸酶P外，研究人员在真核生物中发现了仅由蛋白质构成的核糖核酸酶P，即PRORP。在人类线粒体中，PRORP需与两种蛋白质伴侣结合形成复合物方可发挥功能。在植物中，尽管PRORP酶可单独发挥催化活性，但本研究仍对其互作网络展开探究，以明确其与基因表达通路的整合机制。本研究聚焦拟南芥细胞核内的核糖核酸酶P亚基PRORP2，对其介导的功能互作展开分析。通过免疫亲和策略，本研究证实PRORP2可在体内与tRNA甲基转移酶TRM1A、TRM1B形成复合物。除核糖核酸酶P外，这些酶还可与核糖核酸酶Z（RNase Z）发生相互作用。研究发现TRM1A/B定位于细胞核，且二者的双敲除突变会引发严重的宏观表型。结合免疫检测、质谱分析及转录组-wide tRNA测序（transcriptome-wide tRNAseq）技术，本研究证实TRM1A/B可在体内对70%的胞质tRNA进行26位2,2-二甲基鸟苷（m²,²G26）修饰。本研究借助转录组-wide tRNA测序与RNA印迹杂交技术，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26位带有m²,²G修饰的tRNA——此类tRNA在TRM1A/B突变体中表达量显著下调。综合以上研究结果，催化m²,²G修饰的TRM1A/B可在体内与细胞核核糖核酸酶P功能协同，参与胞质tRNA生物发生的早期步骤。本研究还对拟南芥野生型及TRM1A/B突变体开展了Mim-tRNA测序（Mim-tRNAseq）分析