RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex.

RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.

Fatty acid alpha-dioxygenase from Pisum sativum: temporal and spatial regulation during germination and plant development.

alpha-Dioxygenases are expressed in plants in response to biotic and abiotic stress. They catalyze the enantioselective 2-hydroperoxidation of long-chain fatty acids, the initial step of the alpha-oxidation pathway of fatty acids in plants. In this study, the complete cDNA of an alpha-dioxygenase from germinating pea seeds (Pisum sativum) is presented. The deduced amino acid sequence establishes that the enzyme belongs to the recently characterized family of alpha-dioxygenating enzymes in plants. We also present the first systematic study on the expression of alpha-dioxygenase in germinating and developing pea plants. During germination, alpha-dioxygenase mRNA accumulates in the cotyledons and the embryonic axis of pea seeds de novo. In developing pea plants, the transcript is detected almost exclusively in roots. The accumulation of alpha-dioxygenase protein parallels transcript accumulation in that it is abundant in germinating as well as young plant tissue, and correlates with loss of mRNA during plant maturation. alpha-Dioxygenase enzymatic activity in plant extracts is highest in cotyledons during imbibition. In the embryonic axis and roots of developing plants comparable activity levels are observed, whereas in shoots little alpha-oxidation activity is detected. With this contribution, we present information on the temporal and spatial expression of alpha-dioxygenase during plant germination and development, supporting the hypothesis that the alpha-oxidation pathway of fatty acids plays a role during plant developmental processes.

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids.

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.

Conserved and variable domains within divergent rnase P RNA gene sequences of Prochlorococcus strains.

RNase P RNA gene (rnpB) sequences were PCR-amplified from different members of the Prochlorococcus group. Aligned nucleotide sequences revealed a variance of up to 27% for rnpB. Comparative secondary structure analysis showed that domains P12, P18 and P19 of these novel ribozyme sequences in particular are highly divergent. Thus, these regions in RNase P RNA might serve as potential targets for deoxyoligonucleotide primers for the identification of specific genotypes of Prochlorococcus and for probing field populations. Phylogenetic trees constructed from RNase P RNA sequences were similar to, but not fully congruent with, those derived previously using sequences of the 16S rRNA gene. However, the application of rnpB sequences allowed a better resolution within clades of very closely related genotypes. As is known from 16S rRNA-based phylogenetic trees, sequences from individual strains clustered according to their physiology and the conditions at the original site of isolation, rather than their geographical origin. All sequences obtained from high-light-adapted strains formed a single coherent clade, as did the four sequences from low-light-adapted strains that were previously isolated from the North Atlantic and the subtropical North Pacific. This suggests a remarkable genetic stability of Prochlorococcus genotypes that thrive under identical ecological conditions.