a In vitro RNase P activity assays performed with transcripts corresponding to the TYMV TLS and Arabidopsis pre-tRNACys labeled in 5′ or 3′ as well as CytoRP proteins (cRP), wild type PRORP2 (P2) and a catalytically inactive mutant (CM) as described in the “Methods” section. 0 indicates lanes where no protein was added to the reactions. Numbers on the left indicate the calculated sizes of RNA fragments expected to result from canonical RNase P activity. M shows single-stranded RNA molecular weight markers in nucleotides. The TYMV transcript is 112 nt long, with the final 83 nt being the TLS. RNase P cleavage results in a 92 nt long 5′ product and a 20 nt long 3′ product. Cleavages were performed in at least three independent experiments. b Tridimensional model of CytoRP interaction with the TYMV TLS based on PRORP2 crystal structure13, the TYMV TLS crystal structure14 and both X-ray crystallography and SAXS data on the complex formed by PRORP2 and tRNA13,27. The red star shows the position expected to be cleaved by canonical RNase P activity, close to catalytic aspartates (blue spheres) of the NYN domain. c Precise mapping of the TLS cleavage site by CytoRP was performed by circular RT-PCR of the 5′-cleavage product, cloning, and sequencing. The chromatogram shows the sequence of a representative clone. Arrows and numbers indicate the position and percentage of clones among the 53 clones analyzed resulting from cleavage events. 77% of them took place at the position indicated by the red star in Fig. 2b. d Kinetic analysis of TYMY TLS cleavage by PRORP2 and CytoRP. Experiments were performed with 1 μM protein for 9 reaction times. Experiments were performed with 20, 40, 80, and 120 pM of RNA substrate. Average values from triplicate experiments are shown in a Lineweaver–Burk plot that was used to derive Km and Vmax values. Error bars represent the standard deviation for three replicate experiments. e In vitro RNase P activity assays performed with a transcript corresponding to the ORMV TLS as described for TYMV on a. Numbers on the right indicate the calculated sizes of RNA fragments expected to result from RNase P activity according to a predicted 2D fold of the ORMV TLS presented on the right. Precise mapping of the TLS cleavage site by CytoRP was performed by circular RT-PCR of the 5′-cleavage product, cloning and sequencing. The positions of cleavage sites are indicated on the 2D model, with red arrows and numbers indicate the position and percentage of clones among the 113 clones analyzed resulting from cleavage events (for positions >5%). Source data underlying a, d, and e are provided as a Source Data file.