The MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4) is essential for the accumulation of dicistronic rpl5-cob mRNAs in Arabidopsis thaliana.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3' termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3' end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.

In Arabidopsis thaliana mitochondria 5' end polymorphisms of nad4L-atp4 and nad3-rps12 transcripts are linked to RNA PROCESSING FACTORs 1 and 8.

KEY MESSAGE: RNA PROCESSING FACTORs 1 AND 8 (RPF1 and RPF8), both restorer of fertility like pentatricopeptide repeat proteins, are required for processing of dicistronic nad4L-atp4 and nad3-rps12 transcripts in Arabidopsis mitochondria. In mitochondria of Arabidopsis thaliana (Arabidopsis), the 5' termini of many RNAs are generated on the post-transcriptional level. This process is still poorly understood in terms of both the underlying mechanism as well as proteins required. Our studies now link the generation of polymorphic 5' extremities of the dicistronic nad3-rps12 and nad4L-atp4 transcripts to the function of the P-type pentatricopeptide repeat proteins RNA PROCESSING FACTORs 8 (RPF8) and 1 (RPF1). RPF8 is required to generate the nad3-rps12 -141 5' end in ecotype Van-0 whereas the RPF8 allele in Col has no function in the generation of any 5' terminus of this transcript. This observation strongly suggests the involvement of an additional factor in the generation of the -229 5' end of nad3-rps12 transcripts in Col. RPF1, previously found to be necessary for the generation of the -228 5' end of the major 1538 nucleotide-long nad4 mRNAs, is also important for the formation of nad4L-atp4 transcripts with a 5' end at position -318 in Col. Many Arabidopsis ecotypes contain inactive RPF1 alleles resulting in the accumulation of various low abundant nad4L-atp4 RNAs which might represent precursor and/or degradation products. Some of these ecotypes accumulate major, but slightly smaller RNA species. The introduction of RPF1 into these lines not only establishes the formation of the major nad4L-atp4 dicistronic mRNA with the -318 5' terminus, the presence of this gene also suppresses the accumulation of most alternative nad4L-atp4 RNAs. Beside RPF1, several other factors contribute to nad4L-atp4 transcript formation.

Two NYN domain containing putative nucleases are involved in transcript maturation in Arabidopsis mitochondria.

Plant mitochondrial transcripts frequently undergo maturation processes at their 5' ends. This almost completely enigmatic process requires the function of several proteins such as RNA processing factors, which are selectively involved in distinct 5' processing events. As RNA processing factors represent pentatricopeptide repeat proteins without apparent enzymatic function, it is hypothesized that a ribonuclease, most likely with endonucleolytic activity is involved in the 5' end maturation. We have now applied a reverse genetic approach to analyze the role of two potential mitochondrial nucleases, MNU1 and MNU2, in Arabidopsis thaliana. Both proteins contain several RNA-binding domains and NYN domains found in other endonucleases. A thorough analysis of various mitochondrial transcripts in MNU1 and MNU2 mutants revealed aberrant transcript pattern characterized by a decrease in mature RNA species often accompanied by an accumulation of larger, 5' extended precursor molecules. In addition, severely reduced amounts of nad9 mRNAs in the rpf2-1/mnu2-1 double mutant indicate a corporate function of RNA processing factor 2 and MNU2 in the maturation of these transcripts. However, the dramatic reduction of the nad9 mRNA is not reflected by the level of the corresponding Nad9 protein, which is found to be only moderately lowered. Collectively, our analysis strongly suggests a function of MNU1 and MNU2 in 5' processing of plant mitochondrial transcripts.

In Arabidopsis thaliana distinct alleles encoding mitochondrial RNA PROCESSING FACTOR 4 support the generation of additional 5' termini of ccmB transcripts.

In plant mitochondria, the 5' ends of many transcripts are generated post-transcriptionally. We show that the pentatricopeptide repeat (PPR) protein RNA PROCESSING FACTOR 4 (RPF4) supports the generation of extra 5' ends of ccmB transcripts in Landsberg erecta (Ler) and a number of other Arabidopsis thaliana ecotypes. RPF4 was identified in Ler applying a forward genetic approach supported by complementation studies of ecotype Columbia (Col), which generates the Ler-type extra ccmB 5' termini only after the introduction of the RPF4 allele from Ler. Studies with chimeric RPF4 proteins composed of various parts of the RPF4 proteins from Ler and Col identified differences in the N-terminal and central PPR motifs that explain ecotype-specific variations in ccmB processing. These results fit well with binding site predictions in ccmB transcripts based on the known determinants of nucleotide base recognition by PPR motifs.

In Arabidopsis thaliana two co-adapted cyto-nuclear systems correlate with distinct ccmC transcript sizes.

Plant mitochondrial transcripts undergo maturation processes at both termini. Although frequently observed, the post-transcriptional formation of mature 5' ends is still poorly understood. We now analyzed the processing of transcripts derived from the mitochondrial ccmC gene, coding for a component of the cytochrome c maturation system. In Arabidopsis thaliana (Arabidopsis) there are two mitochondrial ccmC configurations, discriminated by a 66-bp sequence segment located approximately 500 bp upstream of the ccmC gene. In Arabidopsis accessions these divergent mitochondrial genotypes correlate with the generation of two different 5' termini that map to positions around -484 in accession Columbia (Col ccmC genotype) or -390 in accession C24 relative to the translation start codon (C24 ccmC genotype). Previously we identified RNA PROCESSING FACTOR 3 (RPF3), a pentatricopeptide repeat (PPR) protein required for the maturation of ccmCmRNAs with -484 5' ends transcribed from the Col ccmC genotype. Now we identified several accessions defective in maturation of ccmC transcripts. Taking advantage of this natural genetic variation we identified RNA PROCESSING FACTOR 6 (RPF6), a PPR protein necessary for the generation of ccmCmRNAs with -390 5' ends transcribed from the C24 ccmC genotype. Both Col-type and C24-type accessions encode RPF3 and RPF6 so that they can process ccmC transcripts derived from the two different mitochondrial genotypes. These factors and their cognate RNA recognition sites in the different ccmC genotypes are an intriguing example for the evolution of two co-adapted cyto-nuclear systems required for the same process i.e. 5' maturation of ccmC transcripts.

Maturation of 5' ends of plant mitochondrial RNAs.

The generation of mature RNAs, i.e. mRNAs, rRNAs or tRNAs, is a complex process in all genetic systems. RNA-internal processes such as splicing or RNA editing, but also posttranscriptional processes modulating 5' and 3' termini of transcripts, contribute to RNA maturation. In this article, we focus on the posttranscriptional formation of 5' termini of mitochondrial RNAs in seed plants, with particular emphasis on the model plant species Arabidopsis thaliana (Arabidopsis). We will summarize the progress made in recent studies of proteins involved in this process. In addition, we will evaluate whether 5' processing proceeds endo- or exo-nucleolytically. Despite the considerable progress made, many details of this process remain unsolved. In particular, it is still unclear why there is frequent 5' processing of many mRNAs although impaired processing does not interfere with mitochondrial function and plant fitness. Thus, the importance of 5' processing for plant mitochondria is still puzzling.

The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism.

Arabidopsis thaliana possesses six branched-chain aminotransferases (BCAT1-6). Previous studies revealed that some members of this protein family are involved in the biosynthesis of branched-chain amino acids and/or in the Met chain elongation pathway, the initial steps towards the biosynthesis of Met-derived glucosinolates. We now analyzed branched-chain aminotransferase 6 (BCAT6). In vivo GFP-tagging experiments strongly suggest this enzyme to be localized to the cytosol. Substrate specificity assays performed with recombinant enzyme revealed that BCAT6 transaminates Val, Leu and Ile as well as the corresponding 2-oxo acids but also transaminates Met and its cognate ketoacid 4-methyl-2-oxobutanoate. We established single (bcat6-1), double (bcat4-2/bcat6-1) and triple (bcat3-1/bcat4-2/bcat6-1) mutants involving BCAT6 with the latter exhibiting a clear macroscopic phenotype with smaller plants and abnormal leaf morphology. Metabolite profiling of these mutants demonstrated that BCAT6 can contribute to Met chain elongation with the triple mutant line lacking BCAT3, 4 and 6 showing a dramatic reduction of Met-derived glucosinolate species down to 32 and 14% of wild-type levels in plant foliage and seeds, respectively. This drop in glucosinolate levels is accompanied by a 46-fold increase of free Met, demonstrating the important role of the three branched-chain aminotransferases in converting Met to its 2-oxo acid for glucosinolate chain elongation. In addition, we determined the relative amounts of 5'-deoxy-5'-methylthioadenosine, an intermediate of the Met recycling pathway. This metabolite accumulated to relative high amounts in the absence of the cytosolic BCAT4 and BCAT6, suggesting that cytosolic Met salvage also contributes to the biosynthesis of glucosinolates.

RNA Processing Factor 5 is required for efficient 5' cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana.

The 5' ends of many mitochondrial transcripts are generated post-transcriptionally. Recently, we identified three RNA PROCESSING FACTORs required for 5' end maturation of different mitochondrial mRNAs in Arabidopsis thaliana. All of these factors are pentatricopeptide repeat proteins (PPRPs), highly similar to RESTORERs OF FERTILTY (RF), that rescue male fertility in cytoplasmic male-sterile lines from different species. Therefore, we suggested a general role of these RF-like PPRPs in mitochondrial 5' processing. We now identified RNA PROCESSING FACTOR 5, a PPRP not classified as an RF-like protein, required for the efficient 5' maturation of the nad6 and atp9 mRNAs as well as 26S rRNA. The precursor molecules of these RNAs share conserved sequence elements, approximately ranging from positions -50 to +9 relative to mature 5' mRNA termini, suggesting these sequences to be at least part of the cis elements required for processing. The knockout of RPF5 has only a moderate influence on 5' processing of atp9 mRNA, whereas the generation of the mature nad6 mRNA and 26S rRNA is almost completely abolished in the mutant. The latter leads to a 50% decrease of total 26S rRNA species, resulting in an imbalance between the large rRNA and 18S rRNA. Despite these severe changes in RNA levels and in the proportion between the 26S and 18S rRNAs, mitochondrial protein levels appear to be unaltered in the mutant, whereas seed germination capacity is markedly reduced.

RNA processing factor 7 and polynucleotide phosphorylase are necessary for processing and stability of nad2 mRNA in Arabidopsis mitochondria.

Post-transcriptional maturation of plant mitochondrial transcripts requires several steps. Among these, the generation of mature 5' ends is still one of the most enigmatic processes. Toward a characterization of proteins involved in 5' processing of mitochondrial transcripts in Arabidopsis (Arabidopsis thaliana), we now analyzed 5' maturation of nad2 transcripts. Based on natural genetic variation affecting 5' ends of nad2 transcripts in ecotype Can-0 and complementation studies we now identified RNA processing factor 7, which takes part in the generation of the 5' terminus of the mature nad2 mRNA. RPF7 is a relatively short regular P-class pentatricopeptide repeat protein comprising seven canonical P repeats and a single short S repeat. The corresponding allele in Can-0 encodes a truncated version of this protein lacking two C-terminal repeats, which are essential for the function of RPF7. Furthermore we established transgenic plants expressing artifical microRNAs targeting the mitochondrial polynucleotide phosphorylase (PNPase), which results in substantial reduction of the PNPase mRNA levels and strong knockdown of this gene. Detailed quantitative studies of 5' and 3' extended nad2 precursor RNAs in these knockdown plants as well as in the rpf7-1 knockout mutant suggest that 5' processing contributes to the stability of mitochondrial transcripts in plants.

Mitochondrial transcript length polymorphisms are a widespread phenomenon in Arabidopsis thaliana.

Natural genetic variation affects development, physiology, biochemical properties as well as mitochondrial transcripts of the model species Arabidopsis thaliana (Arabidopsis). In a previous study, we identified mitochondrial transcript end polymorphisms in Arabidopsis accessions Columbia, C24 and Landsberg erecta. The polymorphic transcript species could either be assigned to differences in the mitochondrial DNA or to natural genetic variation in the nucleus. To analyze the distribution and to identify additional 5' end polymorphisms we now analyzed 19 mitochondrial transcription units in 26 different accessions. We found additional 5' end polymorphisms indicating that such transcript length differences are a widespread phenomenon in Arabidopsis. The new polymorphisms affect cox1, cox2, nad2 as well nad3-rps12 transcript species. While the cox2 polymorphism can be attributed to a recombination event in the mitochondrial DNA, the nad2 transcript polymorphism is linked to differences in the nuclear DNA. A complex pattern is found for nad3-rps12 mRNA whose 5' ends differ between several accessions. These new polymorphisms provide an important basis for a more detailed characterization of mitochondrial 5' end processing.

RNA PROCESSING FACTOR2 is required for 5' end processing of nad9 and cox3 mRNAs in mitochondria of Arabidopsis thaliana.

In mitochondria of higher plants, the majority of 5' termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 5' end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 5' terminus of transcripts of subunit 9 of the NADH DEHYDROGENASE complex (nad9) and it determines the efficiency of 5' end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 5' processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 5' mRNA processing in mitochondria of higher plants.

P-class pentatricopeptide repeat proteins are required for efficient 5' end formation of plant mitochondrial transcripts.

It is well recognized that flowering plants maintain a particularly broad spectrum of factors to support gene expression in mitochondria. Many of these factors are pentatricopeptide repeat (PPR) proteins that participate in virtually all processes dealing with RNA. One of these processes is the post-transcriptional generation of mature 5' termini of RNA. Several PPR proteins are required for efficient 5' maturation of mitochondrial mRNA and rRNA. These so-called RNA PROCESSING FACTORs (RPF) exclusively represent P-class PPR proteins, mainly composed of canonical PPR motifs without any extra domains. Applying the recent PPR-nucleotide recognition code, binding sites of RPF are predicted on the 5' leader sequences. The sequence-specific interaction of an RPF with one or a few RNA substrates probably directly or indirectly recruits an as-yet-unidentified endonuclease to the processing site(s). The identification and characterization of RPF is a major step toward the understanding of the role of 5' end maturation in flowering plant mitochondria.

A RESTORER OF FERTILITY-like PPR gene is required for 5'-end processing of the nad4 mRNA in mitochondria of Arabidopsis thaliana.

Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.

Complex I-complex II ratio strongly differs in various organs of Arabidopsis thaliana.

In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.

RNA PROCESSING FACTOR3 is crucial for the accumulation of mature ccmC transcripts in mitochondria of Arabidopsis accession Columbia.

RNA PROCESSING FACTOR1 (RPF1) and RPF2 are pentatricopeptide repeat (PPR) proteins involved in 5' processing of different mitochondrial mRNAs in Arabidopsis (Arabidopsis thaliana). Both factors are highly similar to RESTORERS OF FERTILITY (RF), which are part of cytoplasmic male sterility/restoration systems in various plant species. These findings suggest a predominant role of RF-like PPR proteins in posttranscriptional 5' processing. To further explore the functions of this group of proteins, we examined a number of T-DNA lines carrying insertions in the corresponding PPR genes. This screening identified a nearly complete absence of mature ccmC transcripts in an At1g62930 T-DNA insertion line, a phenotype that could be restored by the introduction of the intact At1g62930 gene into the mutant. The insertion in this nuclear gene, which we now call RPF3, also leads to a severe reduction of the CcmC protein in mitochondria. The analysis of C24/rpf3-1 F2 hybrids lacking functional RPF3 genes revealed that this gene has less influence on the generation of the mature ccmC 5' transcript end derived from a distinct ccmC 5' upstream configuration found in mitochondrial DNAs from C24 and other accessions. These data show that a particular function of an RF-like protein is required only in connection with a distinct mtDNA configuration. Our new results further substantiate the fundamental role of RF-like PPR proteins in the posttranscriptional generation of plant mitochondrial 5' transcript termini.

The DEAD-box protein PMH2 is required for efficient group II intron splicing in mitochondria of Arabidopsis thaliana.

In Arabidopsis thaliana the putative mitochondrial RNA helicases PMH1 and PMH2 are members of the large DEAD-box protein family. Our previous characterization of these proteins revealed that PMH1 and/or PMH2 are part of high molecular weight complexes. Now T-DNA insertion lines were established and characterized for each of these genes. Immunodetection analysis of cell suspension cultures established from pmh1-1 and pmh2-1 mutants revealed that indeed both DEAD-box proteins are detectable in large protein complexes with PMH2 being much more abundant than PMH1. In plants the knockout of PMH2 leads to reduced group II intron splicing efficiency. In addition the steady-state levels of several mature mitochondrial mRNAs are decreased while transcription is not influenced. This molecular phenotype suggests that PMH2 acts at the posttranscriptional level with a potential function as RNA chaperone required for formation or maintenance of complex RNA secondary structures of introns rather than a direct role in splicing. In contrast, the investigation of a pmh1-1 knockout line did not reveal any influence of this protein on processing and abundance of mitochondrial transcripts.

In Arabidopsis thaliana Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

In Arabidopsis thaliana, the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates. IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. Here, we investigate the role of the three IPMI SSUs through the analysis of the role of the substrate recognition region spanning five amino acids on the substrate specificity of IPMI SSU1. Furthermore, we analyze in detail the expression pattern of fluorophore-tagged IPMI SSUs throughout plant development. Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. Expression of IPMI SSU1 is spatially separated from the expression of IPMI SSU2 and IPMI SSU3, and IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. Our data show a distinct role for IMPI SSU1 in Leu biosynthesis and for IMPI SSU2 and SSU3 in the Met chain elongation pathway.

Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation.

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit.

Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5' and 3' end formation.

With CR-RT-PCR as primary approach we mapped the 5' and 3' transcript ends of all mitochondrial protein-coding genes in Arabidopsis thaliana. Almost all transcripts analyzed have single major 3' termini, while multiple 5' ends were found for several genes. Some of the identified 5' ends map within promoter motifs suggesting these ends to be derived from transcription initiation while the majority of the 5' termini seems to be generated post-transcriptionally. Assignment of the extremities of 5' leader RNAs revealed clear evidence for an endonucleolytic generation of the major cox1 and atp9 5' mRNA ends. tRNA-like structures, so-called t-elements, are associated either with 5' or with 3' termini of several mRNAs. These secondary structures most likely act as cis-signals for endonucleolytic cleavages by RNase Z and/or RNase P. Since no conserved sequence motif is evident at post-transcriptionally derived ends, we suggest t-elements, stem-loops and probably complex higher order structures as cis-elements for processing. This analysis provides novel insights into 5' and 3' end formation of mRNAs. In addition, the complete transcript map is a substantial and important basis for future studies of gene expression in mitochondria of higher plants.

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells.

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.