A third mitochondrial RNA polymerase in the moss Physcomitrella patens

Abstract

In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.

Fig. 1

Amino acid sequence alignment. Amino acid sequences were compared among Arabidopsis and Physcomitrella RPOT polymerases using the ClustalW algorithm. Sequences with accession numbers: AtRPOTm (CAA69331), AtRPOTp (CAA70210), AtRPOTmp (CAC17120); PpRPOTmp1 (CAC95163); PpRPOTmp2 (CAC95164). Grey boxes indicate conserved blocks in the RPOT polymerase family; functionally crucial residues (McAllister and Raskin ; Sousa et al. 1993) are indicated by asterisks

Fig. 2

ClustalW-derived protein alignment. All three RPOT proteins from Physcomitrella were aligned with SmRPOTm of Selaginella moellendorffii and AtRPOTm of Arabidopsis thaliana using the ClustalW algorithm. Grey arrows below the sequence indicate exons and numbers inside indicate exon numbers. While red arrows mark putative translational start sites, blue boxes highlight intronic insertions in the putative 5′UTRs. Red boxes mark intron positions found in at least one of the Physcomitrella RpoT genes, but lacking in PpRpoT3

Fig. 3

Phylogenetic analysis of plant RPOT proteins. The phylogeny was reconstructed by the PhyML algorithm V3.0 (Guindon et al. 2010) based on a multiple alignment of 54 plant RPOT sequences (listed in Supplementary Table 2) with 100 bootstrap replicates. Prefixes of designations of plant RpoT proteins refer to the abbreviations used in Supplementary Table 2. The tree was rooted with four green algae proteins. Asterisks indicate that experimental evidence for the localization in mitochondria, plastids or both organelles exists. Otherwise, the indicated localization (RPOTm-mitochondrial enzyme, p-plastid enzyme, and mp-enzyme targeted to both mitochondria and plastids) is solely based on the position in the tree. Based on the experimental data shown in Fig. 4, PpRpoT3/PpRPOT3 was renamed into PpRpoTm/PpRPOTm

Fig. 4

Transient expression of GFP fusion proteins in A. thaliana protoplasts. The PpRpoT3 gene fragment encoding a putative transit peptide was inserted into plasmid pOL-GFPS65C to generate a vector driving the expression of utrPpRpoT3 (a) showing mitochondrial GFP localisation; RpoT3 was therefore designated PpRpoTm. Control constructs code for GFP (b, pOL only), mitochondrial CoxIV-GFP (c) and plastidial RecA-GFP (d), respectively. Images were taken by confocal fluorescence microscopy. Scale bar 10 μm

Fig. 5

Proposed localisation of the three phage-type RNA polymerases in Physcomitrella cells. Based on the results of the present study. RPOT3 is localised exclusively to mitochondria and was, therefore, renamed into RPOTm. RPOTmp1 and RPOTmp2 are potentially targeted to both mitochondria and plastids; their localisation might be regulated via usage of different start codons during translation and vary between different tissues