Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.

As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.

Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase.

The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.

Maturation of the E. coli Glu2, Ile1, and Ala1B tRNAs utilizes a complex processing pathway.

Despite significant progress in understanding the diversity of tRNA processing pathways in Escherichia coli, the mechanism for the maturation of tRNAs encoded within the rRNA operons has not received much attention. Here, we show that the Glu2, Ile1, and Ala1B tRNAs, encoded by 10 genes located between the 16S and 23S rRNAs in the seven rRNA operons, are matured via a RNase E-independent processing pathway that utilizes at least six different enzymes. It has been shown that the Glu2 and Ile1-Ala1B pre-tRNAs released by initial RNase III cleavages of the 30S primary rRNA transcripts retain extended 5'-leader (35-139 nt) and 3'-trailer (166-185 nt) sequences. However, the 5' maturation of the tRNAs by RNase P is inhibited until the trailer sequences are shortened to 1-4 nucleotides, initially by a second RNase III cleavage at 31-42 nucleotides downstream of the CCA determinant followed by exonucleolytic trimming. The RNase III cleaved Glu2 and Ile1-Ala1B trailer fragments are degraded via PAP I- dependent exonucleolytic decay. Compared to the six previously characterized tRNA processing pathways, maturation of the Glu2, Ile1, and Ala1B tRNAs is considerably more complex and appears to be distinct from what occurs in Gram-positive bacteria.

The absence of PNPase activity in Enterococcus faecalis results in alterations of the bacterial cell-wall but induces high proteolytic and adhesion activities.

Enterococci are lactic acid bacteria (LAB) used as starters and probiotics, delineating their positive attributes. Nevertheless, enterococci can be culprit for thousands of infectious diseases, including urinary tract infections, bacteremia and endocarditis. Here, we aim to determine the impact of polynucleotide phosphorylase (PNPase) in the biology of Enterococcus faecalis 14; a human isolate from meconium. Thus, a mutant strain deficient in PNPase synthesis, named ΔpnpA mutant, was genetically obtained. After that, a transcriptomic study revealed a set of 244 genes differentially expressed in the ΔpnpA mutant compared with the wild-type strain, when exploiting RNAs extracted from these strains after 3 and 6 h of growth. Differentially expressed genes include those involved in cell wall synthesis, adhesion, biofilm formation, bacterial competence and conjugation, stress response, transport, DNA repair and many other functions related to the primary and secondary metabolism of the bacteria. Moreover, the ΔpnpA mutant showed an altered cell envelope ultrastructure compared with the WT strain, and is also distinguished by a strong adhesion capacity on eukaryotic cell as well as a high proteolytic activity. This study, which combines genetics, physiology and transcriptomics enabled us to show further biological functions that could be directly or indirectly controlled by the PNPase in E. faecalis 14.

Pivotal Roles for Ribonucleases in Streptococcus pneumoniae Pathogenesis.

RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis, as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that the abundances of nearly 200 mRNA transcripts were significantly increased, whereas those of several pneumococcal small regulatory RNAs (sRNAs), including the Ccn (CiaR-controlled noncoding RNA) sRNAs, were altered in the Δrny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Δpnp mutants showed no growth defect in vitro but differentially expressed a total of 40 transcripts, including the tryptophan biosynthesis operon genes and numerous 5' cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Together, our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase mediates virulence phenotypes, likely through sRNA regulation. IMPORTANCE Streptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. RNases are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes, including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled noncoding (Ccn) sRNAs.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

Pseudomonas aeruginosa Polynucleotide Phosphorylase Controls Tolerance to Aminoglycoside Antibiotics by Regulating the MexXY Multidrug Efflux Pump.

Pseudomonas aeruginosa is an opportunistic pathogen that shows high intrinsic resistance to a variety of antibiotics. The MexX-MexY-OprM efflux pump plays an important role in bacterial resistance to aminoglycoside antibiotics. Polynucleotide phosphorylase (PNPase) is a highly conserved exonuclease that plays important roles in RNA processing and the bacterial response to environmental stresses. Previously, we demonstrated that PNPase controls the tolerance to fluoroquinolone antibiotics by influencing the production of pyocin in P. aeruginosa In this study, we found that mutation of the PNPase-encoding gene (pnp) in P. aeruginosa increases bacterial tolerance to aminoglycoside antibiotics. We further demonstrate that the upregulation of the mexXY genes is responsible for the increased tolerance of the pnp mutant. Furthermore, our experimental results revealed that PNPase controls the translation of the armZ mRNA through its 5' untranslated region (UTR). ArmZ had previously been shown to positively regulate the expression of mexXY Therefore, our results revealed a novel role of PNPase in the regulation of armZ and subsequently the MexXY efflux pump.

The Bacterial Ro60 Protein and Its Noncoding Y RNA Regulators.

Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. In D. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.

Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding.

The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δpnp with plasmids expressing mutant PNPases caused increased cell tolerance to H2O2. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.

Phylogeny and Evolution of RNA 3'-Nucleotidyltransferases in Bacteria.

The tRNA nucleotidyltransferases and poly(A) polymerases belong to a superfamily of nucleotidyltransferases. The amino acid sequences of a number of bacterial tRNA nucleotidyltransferases and poly(A) polymerases have been used to construct a rooted, neighbor-joining phylogenetic tree. Using information gleaned from that analysis, along with data from the rRNA-based phylogenetic tree, structural data available on a number of members of the superfamily and other biochemical information on the superfamily, it is possible to suggest a scheme for the evolution of the bacterial tRNA nucleotidyltransferases and poly(A) polymerases from ancestral species. Elements of that scheme are discussed along with questions arising from the scheme which can be explored experimentally.

Pseudomonas aeruginosa Polynucleotide Phosphorylase Contributes to Ciprofloxacin Resistance by Regulating PrtR.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3'-5' exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5' untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa.

Impact on strain growth and butenyl-spinosyn biosynthesis by overexpression of polynucleotide phosphorylase gene in Saccharopolyspora pogona.

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement.

Novel Aspects of Polynucleotide Phosphorylase Function in Streptomyces.

Polynucleotide phosphorylase (PNPase) is a 3'-5'-exoribnuclease that is found in most bacteria and in some eukaryotic organelles. The enzyme plays a key role in RNA decay in these systems. PNPase structure and function have been studied extensively in Escherichiacoli, but there are several important aspects of PNPase function in Streptomyces that differ from what is observed in E. coli and other bacterial genera. This review highlights several of those differences: (1) the organization and expression of the PNPase gene in Streptomyces; (2) the possible function of PNPase as an RNA 3'-polyribonucleotide polymerase in Streptomyces; (3) the function of PNPase as both an exoribonuclease and as an RNA 3'-polyribonucleotide polymerase in Streptomyces; (4) the function of (p)ppGpp as a PNPase effector in Streptomyces. The review concludes with a consideration of a number of unanswered questions regarding the function of Streptomyces PNPase, which can be examined experimentally.

A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins.

There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a computational protocol for the characterization of interactions between proteins and RNA containing post-transcriptional modifications. Given an RNA-protein complex structure, potential RNA modified ribonucleoside positions, and molecular mechanics parameters for capturing energetics of RNA modifications, our protocol operates in two stages. In the first stage, a decision-making tool, comprising short simulations and interaction energy calculations, performs a fast and efficient search in a high-throughput fashion, through a list of different types of RNA modifications categorized into trees according to their structural and physicochemical properties, and selects a subset of RNA modifications prone to interact with the target protein. In the second stage, RNA modifications that are selected as recognized by the protein are examined in-detail using all-atom simulations and free energy calculations. We implement and experimentally validate this protocol in a test case involving the study of RNA modifications in complex with Escherichia coli (E. coli) protein Polynucleotide Phosphorylase (PNPase), depicting the favorable interaction between 8-oxo-7,8-dihydroguanosine (8-oxoG) RNA modification and PNPase. Further advancement of the protocol can broaden our understanding of protein interactions with all known RNA modifications in several systems.

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis.

Bacillus subtilis is a wealth source of lipopeptide molecules such as iturins, surfactins and fengycins or plipastatins endowed with a range of biological activities. These molecules, designated secondary metabolites, are synthesized via non-ribosomal peptides synthesis (NRPS) machinery and are most often subjected to a complex regulation with involvement of several regulatory factors. To gain novel insights on mechanism regulating fengycin production, we investigated the effect of the fascinating polynucleotide phosphorylase (PNPase), as well as the effect of lipopeptide surfactin. Compared to the wild type, the production of fengycin in the mutant strains B. subtilis BBG235 and BBG236 altered for PNPase has not only decreased to about 70 and 40%, respectively, but also hampered its antifungal activity towards the plant pathogen Botrytis cinerea. On the other hand, mutant strains BBG231 (srfAA-) and BBG232 (srfAC-) displayed different levels of fengycin production. BBG231 had registered an important decrease in fengycin production, comparable to that observed for BBG235 or BBG236. This study permitted to establish that the products of pnpA gene (PNPase), and srfAA- (surfactin synthetase) are involved in fengycin production.

Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli.

BACKGROUND: Polynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes. RESULTS: In this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover. CONCLUSIONS: Our data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

Regulation of mRNA Decay in Bacteria.

Gram-negative and gram-positive bacteria use a variety of enzymatic pathways to degrade mRNAs. Although several recent reviews have outlined these pathways, much less attention has been paid to the regulation of mRNA decay. The functional half-life of a particular mRNA, which affects how much protein is synthesized from it, is determined by a combination of multiple factors. These include, but are not necessarily limited to, (a) stability elements at either the 5' or the 3' terminus, (b) posttranscriptional modifications, (c) ribosome density on individual mRNAs, (d) small regulatory RNA (sRNA) interactions with mRNAs, (e) regulatory proteins that alter ribonuclease binding affinities, (f) the presence or absence of endonucleolytic cleavage sites, (g) control of intracellular ribonuclease levels, and (h) physical location within the cell. Changes in physiological conditions associated with environmental alterations can significantly alter the impact of these factors in the decay of a particular mRNA.

The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes.

In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3' ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.

Sm-like protein Hfq: Composition of the native complex, modifications, and interactions.

The bacterial Sm-like protein Hfq has been linked functionally to reactions that involve RNA; however, its explicit role and primary cellular localization remain elusive. We carried out a detailed biochemical characterization of native Escherichia coli Hfq obtained through methods that preserve its posttranslational modifications. ESI-MS analyses indicate modifications in 2-3 subunits/hexamer with a molecular mass matching that of an oxidized C:18 lipid. We show that the majority of cellular Hfq cannot be extracted without detergents and that purified Hfq can be retained on hydrophobic matrices. Analyses of purified Hfq and the native Hfq complexes observed in whole-cell E. coli extracts indicate the existence of dodecameric assemblies likely stabilized by interlocking C-terminal polypeptides originating from separate Hfq hexamers and/or accessory nucleic acid. We demonstrate that cellular Hfq is redistributed between transcription complexes and an insoluble fraction that includes protein complexes harboring polynucleotide phosphorylase (PNP). This distribution pattern is consistent with a function at the interface of the apparatuses responsible for synthesis and degradation of RNA. Taken together with the results of prior studies, these results suggest that Hfq could function as an anchor/coupling factor responsible for de-solubilization of RNA and its tethering to the degradosome complex.

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.