A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (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 identify potential tRNA maturation complexes. 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 TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 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/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

CoLoC-seq probes the global topology of organelle transcriptomes.

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

How RNases Shape Mitochondrial Transcriptomes.

Mitochondria are the power houses of eukaryote cells. These endosymbiotic organelles of prokaryote origin are considered as semi-autonomous since they have retained a genome and fully functional gene expression mechanisms. These pathways are particularly interesting because they combine features inherited from the bacterial ancestor of mitochondria with characteristics that appeared during eukaryote evolution. RNA biology is thus particularly diverse in mitochondria. It involves an unexpectedly vast array of factors, some of which being universal to all mitochondria and others being specific from specific eukaryote clades. Among them, ribonucleases are particularly prominent. They play pivotal functions such as the maturation of transcript ends, RNA degradation and surveillance functions that are required to attain the pool of mature RNAs required to synthesize essential mitochondrial proteins such as respiratory chain proteins. Beyond these functions, mitochondrial ribonucleases are also involved in the maintenance and replication of mitochondrial DNA, and even possibly in the biogenesis of mitochondrial ribosomes. The diversity of mitochondrial RNases is reviewed here, showing for instance how in some cases a bacterial-type enzyme was kept in some eukaryotes, while in other clades, eukaryote specific enzymes were recruited for the same function.