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The mitochondrial transcriptome of the true acellular slime mold Physarum polycephalum (Physarum) undergoes extensive RNA editing to produce precise, site-specific changes not encoded at the DNA level. RNA editing in Physarum is essential for proper mitochondrial gene expression by creating open reading frames in protein-coding RNAs and by altering the folding stability of structural RNAs. Physarum carries out one of the widest range of RNA editing events yet described. These changes to mitochondrial RNAs involve the site-specific insertion of over 1300 “extra” nucleotides, deletion of 3 encoded nucleotides, targeted base conversions, and the removal and replacement of nucleotides at the 5′ end of certain tRNAs. While these sequence alterations are absolutely required for the production of functional transcripts, it remains a mystery why they are not encoded in the mitochondrial genome. Although various examples of RNA editing have been described in several eukaryotic organisms, Physarum mitochondria achieve non-templated nucleotide insertion by a unique co-transcriptional mechanism. The cis-acting elements required for insertion editing have been localized to a relatively small region in the vicinity of editing sites, but the details as to how editing sites are recognized and the identity of the trans-acting editing factor(s) required for insertion of these extra nucleotides remain to be elucidated. Two other mechanistically distinct forms of editing, 5′ tRNA editing and C-to-U conversion, have also been described, which proceed via two independent, posttranscriptional pathways. The relatively recent availability of genome and transcriptome sequence data has facilitated the identification of potential candidates for each of these activities and experiments to determine which of these factors are involved in the various forms of editing are underway.

The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These “extra” nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

The tRNAHis guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3′-to-5′ nucleotide addition reaction that specifies tRNAHis identity in eukaryotes, the discovery of a generalized base pair–dependent 3′-to-5′ polymerase reaction greatly expanded the scope of Thg1 family–catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3′-to-5′ polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ∼30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family–catalyzed 3′-to-5′ addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.

Genes with sequence similarity to the yeast tRNAHis guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNAHis maturation to 5′-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3′-5′ nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G−1 addition enzyme likely to be responsible for tRNAHis maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5′-tRNA editing, based on their ability to efficiently repair the 5′ ends of mitochondrial tRNA editing substrates. Although 5′-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5′-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3′-5′ nucleotide addition reactions for diverse biological functions in the same organism.

The mitochondrial genome of Physarum polycephalum encodes five tRNAs, four of which are edited by nucleotide insertion. Two of these tRNAs, tRNAmet1 and tRNAmet2, contain predicted mismatches at the beginning (proximal end) of the acceptor stem. In addition, the putative 5′ end of tRNAmet2 overlaps the 3′ end of a small, abundant, noncoding RNA, which we term ppoRNA. These anomalies led us to hypothesize that these two Physarum mitochondrial tRNAs undergo additional editing events. Here, we show that tRNAmet1 and tRNAmet2 each has a nonencoded G at its 5′ end. In contrast to the other nucleotides that are added to Physarum mitochondrial RNAs, these extra G residues are likely added post-transcriptionally based on (1) the absence of added G in precursor transcripts containing inserted C and AA residues, (2) the presence of potential intermediates characteristic of 5′ replacement editing, and (3) preferential incorporation of GTP into tRNA molecules under conditions that do not support transcription. This is the first report of both post-transcriptional nucleotide insertions and the addition of single Gs in P. polycephalum mitochondrial transcripts. We postulate that tRNAmet1 and tRNAmet2 are acted upon by an activity similar to that present in the mitochondria of certain other amoebozoons and chytrid fungi, suggesting that enzymes that repair the 5′ end of tRNAs may be widespread.

In promoter DNA, the preferred distance of the -10 and -35 elements for interacting with RNA polymerase-bound sigma(70) is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C-terminal domain of sigma(70), including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing sigma(70).

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental\ud model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is\ud unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the\ud genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with\ud simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases.\ud Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into\ud earlyeukaryoteevolution.Wedescribeextensiveuseofhistidinekinase-basedtwo-componentsystemsandtyrosinekinasesignaling,\ud the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide\ud repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes.\ud Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of\ud Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases inAcanthamoeba and Physarum\ud as representatives of two distantly related subdivisions ofAmoebozoa argues against the later emergence of tyrosine kinase signaling\ud in the opisthokont lineage and also against the acquisition by horizontal gene transfer

Gene finding is complicated in organisms that exhibit insertional RNA editing. Here, we demonstrate how our new algorithm Predictor of Insertional Editing (PIE) can be used to locate genes whose mRNAs are subjected to multiple frameshifting events, and extend the algorithm to include probabilistic predictions for sites of nucleotide insertion; this feature is particularly useful when designing primers for sequencing edited RNAs. Applying this algorithm, we successfully identified the nad2, nad4L, nad6 and atp8 genes within the mitochondrial genome of Physarum polycephalum, which had gone undetected by existing programs. Characterization of their mRNA products led to the unanticipated discovery of nucleotide deletion editing in Physarum. The deletion event, which results in the removal of three adjacent A residues, was confirmed by primer extension sequencing of total RNA. This finding is remarkable in that it comprises the first known instance of nucleotide deletion in this organelle, to be contrasted with nearly 500 sites of single and dinucleotide addition in characterized mitochondrial RNAs. Statistical analysis of this larger pool of editing sites indicates that there are significant biases in the 2 nt immediately upstream of editing sites, including a reduced incidence of nucleotide repeats, in addition to the previously identified purine-U bias.

Many of the RNAs transcribed from the mitochondrial genome of Physarum polycephalum are edited by the insertion of nonencoded nucleotides, which are added either singly or as dinucleotides. In addition, at least one mRNA is also subject to substitutional editing in which encoded C residues are changed to U residues posttranscriptionally. We have shown previously that the predominant type of editing in these organelles, the insertion of nonencoded single C residues, occurs cotranscriptionally at the growing end of the RNA chain. However, less is known about the timing of dinucleotide addition, and it has been suggested that these insertions occur at a later stage in RNA maturation. Here we examine the addition of both single nucleotides and dinucleotides into nascent RNAs synthesized in vitro and in vivo. The distribution of added nucleotides within individual cloned cDNAs supports the hypothesis that all insertion sites are processed at the same time relative to transcription. In addition, the patterns of partial editing and misediting observed within these nascent RNAs suggest that separate factors may be required at a subset of dinucleotide insertion sites and raise the possibility that in vivo, nucleotides may be added to RNA and then changed posttranscriptionally.

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of non-encoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.

In vitro approaches have facilitated mechanistic studies designed to look at the timing, accuracy and efficiency of insertional editing of newly synthesized RNAs. Because different assays are required for these distinct RNA populations, the two approaches are discussed separately. Importantly, upon incubation under editing conditions, nucleotide insertion into previously synthesized, unedited RNA was not observed, even though newly transcribed RNA made during the ‘’chase’’ reaction was fully edited . The lack of unedited regions in RNAs labeled in isolated mitochondria under standard, non-limiting nucleotide concentrations and the proximity of RNA editing to the site of transcription, coupled with the inability of the editing activity to insert nucleotides into previously synthesized RNAs, argue that there is a limited ‘’window of opportunity’’ for editing in Physarum mitochondria. Given the proximity of nucleotide insertion to the active site of transcription, it is likely that the Physarum mitochondrial RNA polymerase plays some role, direct or indirect, in the editing process. Nonencoded nucleotides are known to be added very close to the growing end of the RNA chain in Physarum mitochondria , and it is possible that they are added concurrently with RNA synthesis. Although transcription normally is templated, many RNA polymerases are capable of adding nucleotides in a nontemplated or pseudotemplated manner. Much of the mitochondrial genome of Physarum has yet to be sequenced, and it is possible that additional types of editing events may yet be discovered upon characterization of the remaining genes and their RNA products.

▪ Abstract RNA editing can be broadly defined as any site-specific alteration in an RNA sequence that could have been copied from the template, excluding changes due to processes such as RNA splicing and polyadenylation. Changes in gene expression attributed to editing have been described in organisms from unicellular protozoa to man, and can affect the mRNAs, tRNAs, and rRNAs present in all cellular compartments. These sequence revisions, which include both the insertion and deletion of nucleotides, and the conversion of one base to another, involve a wide range of largely unrelated mechanisms. Recent advances in the development of in vitro editing and transgenic systems for these varied modifications have provided a better understanding of similarities and differences between the biochemical strategies, regulatory sequences, and cellular factors responsible for such RNA processing events.

The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.

Maturation of Physarum mitochondrial RNA involves the highly specific insertion of nonencoded nucleotides at multiple locations. To investigate the mechanism(s) by which this occurs, we previously developed an isolated mitochondrial system in which run-on transcripts are accurately and efficiently edited by nucleotide insertion. Here we show that under limiting concentrations of exogenous nucleotides the mitochondrial RNA polymerases stall, generating a population of nascent RNAs that can be extended upon addition of limiting nucleotide. Several of these RNA species have been characterized and were found to be fully edited, indicating that nascent RNA is a substrate for nucleotide insertion in isolated Physarum mitochondria. Remarkably, these RNAs are edited at positions located within 14–22 nucleotides of the polymerase active site, suggesting that insertional editing may be physically or functionally associated with transcription. The absence of unedited RNA in these experiments indicates that large tracts of RNA downstream of editing sites are not required for nucleotide addition, and argues that insertional editing in Physarum occurs with a 5′ to 3′ polarity. These data also provide strong evidence that insertional editing in Physarum is mechanistically distinct from editing in kinetoplastid systems.

Deoxyribonucleic acid (DNA) is often described as a “blueprint for life,” implying that knowledge of the primary sequence of nucleic acids in a genome can give biologists a complete picture of the organism built from these plans. However, neither life nor DNA is that simple. In reality, the sequence of nucleotides in a genome is often just a starting point for the construction of DNA genes and ribonucleic acid (RNA) transcripts, which undergo many alterations before organisms actually use the information to fashion their building materials.

The three-dimensional conformation of a 24-nucleotide variant of the RNA binding sequence for the coat protein of bacteriophage R17 has been analyzed using NMR, molecular dynamics, and energy minimization. The imino proton spectrum is consistent with base pairing requirements for coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including deconvolution and path analysis methods. An average of 21 distance constraints per residue were used in molecular dynamics calculations to obtain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to C15 in the upper stem and loop appears to be flexible. Several of these residues have a large fraction of S-puckered ribose rings, rather than the N-forms characteristic of RNA duplexes. There is considerable variation in the low-energy loop conformations that satisfy the distance constraints at this preliminary level of refinement. The Shine-Dalgarno ribosome binding site is exposed, and only two apparently weak base pairs would have to break for the 16S ribosomal RNA to bind and the ribosome to initiate translation of the replicase gene. Although the loop form must be regarded as tentative, the known interaction sites with the coat protein are easily accessible from the major groove side of the loop.

Ribonucleic acid (RNA) editing can be broadly defined as any process that changes the sequence of an RNA molecule in a way that could be encoded within the genome. These small, targeted changes at the RNA level can have profound effects on gene expression. Such programmed alterations can be controlled in a tissue-specific or developmentally regulated fashion, providing an alternative means of augmenting the information encoded within the genome. In mammalian cells, cytidine to uridine (C to U) and adenosine to inosine (A to I) changes have been observed in both coding and noncoding sequences, including microRNAs, and dysregulation has been linked to disease states. Editing enzymes are often essential, and can act on viral, as well as cellular RNAs. Some human pathogens utilise additional forms of RNA editing for gene expression; these forms of editing provide potential therapeutic targets. Key Concepts: RNA editing is a means of generating genetic diversity that is far more flexible than simply encoding information at the genomic level. Editing is regulated both developmentally and spatially, and dysregulation can have serious consequences. Most editing events in humans fall outside of coding regions; the functional consequences of many of these changes are still being elucidated, but will likely be far-reaching. Editing can impact the function of noncoding RNAs, including those involved in RNA silencing. RNA editing can have both antiviral and proviral effects. Keywords: base substitutions; gene expression; nucleotide insertion; modification; RNA editing

RNAs transcribed from the mitochondrial genome of Physarum polycephalum are heavily edited. The most prevalent editing event is the insertion of single Cs, with Us and dinucleotides also added at specific sites. The existence of insertional editing makes gene identification difficult and localization of editing sites has relied upon characterization of individual cDNAs. We have now determined the complete mitochondrial transcriptome of Physarum using Illumina deep sequencing of purified mitochondrial RNA. We report the first instances of A and G insertions and sites of partial and extragenic editing in Physarum mitochondrial RNAs, as well as an additional 772 C, U and dinucleotide insertions. The notable lack of antisense RNAs in our non-size selected, directional library argues strongly against an RNA-guided editing mechanism. Also of interest are our findings that sites of C to U changes are unedited at a significantly higher frequency than insertional editing sites and that substitutional editing of neighboring sites appears to be coupled. Finally, in addition to the characterization of RNAs from 17 predicted genes, our data identified nine new mitochondrial genes, four of which encode proteins that do not resemble other proteins in the database. Curiously, one of the latter mRNAs contains no editing sites.

The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.

The term RNA editing encompasses two types of specific alterations in the coding potential of RNA molecules: base substitution and the insertion (or deletion) of nucleotides. Such changes in RNA sequence can have profound effects on gene expression, and, indeed, most genes in the mitochondria of plants, trypanosomatids, and Physarum appear to require editing for their expression. We describe here the first instance of the utilization of both types of RNA editing in the processing of a single mRNA, that of the mitochondrially encoded cytochrome oxidase subunit I of the acellular slime mold, Physarum polycephalum. Editing of this mRNA includes the insertion of cytidine, guanosine, and uridine residues, as well as the apparent conversion of cytidines to uridines. No edited version of this gene was detected in Physarum DNA, and amino acid alignments suggest that both types of RNA editing are required to produce a functional protein.

5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii , a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum . These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes , we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium . Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.

The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU)-substituted RNA using medium-wavelength UV light. The authors have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment. Formation of the covalent complex is dependent upon the presence of BrU at position {minus}5 of the RNA and specific binding of the RNA by coat protein. The amount of cross-linking increases with time and depends on the light source and conditions used. Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively. The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003. While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed.

Motivation: Insertional RNA editing renders gene prediction very difficult compared to organisms without such RNA editing. A case in point is the mitochondrial genome of Physarum polycephalum in which only about one-third of the number of genes that are to be expected given its length are annotated. Thus, gene prediction methods that explicitly take into account insertional editing are needed for successful annotation of such genomes. Results: We annotate the mitochondrial genome of P.polycephalum using several different approaches for gene prediction in organisms with insertional RNA editing. We computationally validate our annotations by comparing the results from different methods against each other and as proof of concept experimentally validate two of the newly predicted genes. We more than double the number of annotated putative genes in this organism and find several intriguing candidate genes that are not expected in a mitochondrial genome. Availability: The C source code of the programs described here are available upon request from the corresponding author. Contact: bundschuh@mps.ohio-state.edu

Mitochondrial gene expression in the acellular slime mold Physarum polycephalum requires a diverse array of RNA editing events. Virtually all transcripts encoded in the organelle are subject to changes at the RNA level, including both mRNAs and stable RNAs. Roughly 500 editing events involving nucleotide insertion or deletion have been confirmed thus far; these occur co-transcriptionally. Base changes also occur in Physarum mitochondria, but these are much rarer and occur post-transcriptionally. This chapter focuses on the experimental approaches used to dissect the unique mechanism of insertion/deletion editing in Physarum mitochondria.

Mitochondrial RNAs in the myxomycete Physarum polycephalum differ from the templates from which they are transcribed in defined ways. Most transcripts contain nucleotides that are not present in their respective genes. These "extra" nucleotides are added during RNA synthesis by an unknown mechanism. Other differences observed between Physarum mitochondrial RNAs and the mitochondrial genome include nucleotide deletions, C to U changes, and the replacement of one nucleotide for another at the 5' end of tRNAs. All of these alterations are remarkably precise and highly efficient in vivo. Many of these editing events can be replicated in vitro, and here we describe both the in vitro systems used to study editing in Physarum mitochondria and the assays that have been developed to assess the extent of editing of RNAs generated in these systems at individual sites.

Mitochondrial RNAs in the myxomycete Physarum polycephalum differ from the templates from which they are transcribed in defined ways. Most transcripts contain nucleotides that are not present in their respective genes. These “extra” nucleotides are added during RNA synthesis by an unknown mechanism. Other differences observed between Physarum mitochondrial RNAs and the mitochondrial genome include nucleotide deletions, C to U changes, and the replacement of one nucleotide for another at the 5′ end of tRNAs. All of these alterations are remarkably precise and highly efficient in vivo . Many of these editing events can be replicated in vitro , and here we describe both the in vitro systems used to study editing in Physarum mitochondria and the assays that have been developed to assess the extent of editing of RNAs generated in these systems at individual sites.

RNA editing, which results in the creation of RNA molecules that differ from the template from which they were made, is a highly specific process. Alterations include converting one base to another, removal of one nucleotide and substitution of another, deletion of encoded residues, and insertion of non-templated nucleotides. Such changes have marked effects on gene expression, ranging from defined amino acid changes to the de novo creation of entire open reading frames. Editing can be regulated in a developmental or tissue-specific manner, and is likely to play a role in the etiology of human disease. To cite this article: J.M. Gott, C. R. Biologies 326 (2003).

Part I. RNA Interference and Gene Silencing RNA Interference: Historical Overview and Significance Mary K. Montgomery Delivery of Double-Stranded RNA into Caenorhabditis elegans Dawn Hull and Lisa Timmons Induction and Biochemical Purification of RNA-Induced Silencing Complex From Drosophila S2 Cells Amy A. Caudy and Gregory J. Hannon Analysis of Gene Function in Trypanosoma brucei Using RNA Interference Appolinaire Djikeng, Shuiyuan Shen, Christian Tschudi, and Elisabetta Ullu Short Hairpin Activated Gene Silencing in Mammalian Cells Patrick J. Paddison, Amy A. Caudy, Ravi Sachidanandam, and Gregory J. Hannon Geminivirus Vectors for Transient Gene Silencing in Plants Nooduan Muangsan and Dominique Robertson Posttranscriptional Gene Silencing in Plants Susan Varsha Wesley, Chris Helliwell, Ming-Bo Wang, and Peter Waterhouse Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning Victor Ambros and Rosalind C. Lee Part II. RNA Editing and Modification A Historical Perspective on RNA Editing: How the Peculiar and Bizarre Became Mainstream Donna J. Koslowsky Identification of Substrates for Adenosine Deaminases That Act on RNA Daniel P. Morse Purification and Assay of Recombinant ADAR Proteins Expressed in the Yeast Pichia pastoris or in Escherichia coli Gillian M. Ring, Mary A. O'Connell, and Liam P. Keegan Isolation of an mRNA-Binding Protein Involved in C-to-U Editing Carri A. Gerber, Anne Relich, and Donna M. Driscoll In Vitro Assays for Kinetoplastid U Insertion-Deletion Editing and Associated Activities Kenneth Stuart, Reza Salavati, Robert P. Igo, Jr., Nancy Lewis Ernst, Setareh S. Palazzo, and Bingbing Wang Identification and Characterization of Trypanosome RNA-Editing Complex Components Kenneth Stuart, Aswini K.Panigrahi, and Achim Schnaufer Chimeric Templates and Assays Used to Study Physarum Cotranscriptional Insertional Editing In Vitro Elaine M. Byrne Methods for Analysis of Mitochondrial tRNA Editing in Acanthamoeba castellanii Amanda J. Lohan and Michael W. Gray In Vitro RNA Editing Systems From Higher Plant Chloroplasts Tetsuro Hirose, Tetsuya Miyamoto, Junichi Obokata, and Masahiro Sugiura Studying RNA Editing in Transgenic Chloroplasts of Higher Plants Ralph Bock Detection and Quantification of Modified Nucleotides in RNA Using Thin-Layer Chromatography Henri Grosjean, Gerard Keith, and Louis Droogmans Functional Characterization of 2'-O-Methylation and Pseudouridylation Guide RNAs Tamas Kiss and Beata E. Jady Experimental RNomics: A Global Approach to Identifying Small Nuclear RNAs and Their Targets in Different Model Organisms Alexander Huttenhofer, Jerome Cavaille, and Jean-Pierre Bachellerie Index

Gene expression in the mitochondria of kinetoplastid protists like the human pathogen Trypanosoma brucei occurs via a remarkable pathway involving both the deletion of encoded uridines (Us) and the insertion of extra Us at defined positions within mRNAs synthesized from “cryptogenes” in the maxicircle DNA (see refs. 1 and 2 for recent reviews). In some cases, >50% of the nucleotides present in the mature mRNA are generated by RNA editing events. U addition and removal create sense from nonsense, producing ORFs that encode the proteins involved in oxidative phosphorylation and other mitochondrial functions. Many of these changes are developmentally regulated, occurring only in the procyclic (insect) stage or the bloodstream (mammalian) form. Although the overall mechanism by which this extraordinary phenomenon occurs has been sketched out (3–6), filling in the details has been difficult because of the low abundance and complexity of the editing apparatus, the low efficiency of in vitro editing assays, the lack of assays for processive editing, and difficulties in assigning functions to specific proteins. The article by Aphasizhev et al. (7) in this issue of PNAS provides important information regarding the function of two of the key enzymes involved in this fascinating process. The insertion and deletion of Us into kinetoplast mRNAs occur through the concerted action of a series of enzymes (Fig. 1) (see references in ref. 2). Specificity is provided by small RNA molecules termed guide RNAs (gRNAs), which base-pair to preedited mRNAs just downstream of editing sites (8). gRNAs have three functional domains: an anchor region, which anneals to the substrate; a guiding region, which directs the insertion or deletion of U residues; and an oligo(U) tail, which is added posttranscriptionally and is thought to help tether the purine-rich …

Publisher Summary The flow of genetic information from DNA to protein requires a mRNA intermediate. In all eukaryotes, mature mRNAs are not simple copies of the DNA sequence. Rather, mRNAs are transcribed by RNA polymerase as precursor molecules that must be processed prior to translation. mRNA processing is complex and includes 5′ end-maturation, 3′ end-maturation and removal of non-coding sequences via splicing. These fundamental steps of RNA processing are shared between parasitic organisms and their hosts. Remarkably, the analysis of parasite gene expression has revealed novel and unusual types of mRNA processing reactions. The two best understood examples of such reactions are trans-splicing and RNA editing. The purpose of this chapter is to provide a discussion of our current understanding of these two mRNA maturation pathways. . The chapter explains that mitochondrial function is drastically reduced in bloodstream parasites and many aspects of RNA metabolism are developmentally regulated. And because it is currently unknown whether partially edited mRNAs are translated, the potential also exists for the production of different protein isoforms during development.

Insertional RNA editing in Physarum polycephalum is a complex process involving the specific addition of non-templated nucleotides to nascent mitochondrial transcripts. Since all four ribonucleotides are substrates for the editing activity(s), both the site of insertion and the identity of the nucleotide to be added at a particular position must be specified, but the signals for these events have yet to be elucidated. Here we report the occurrence of sporadic errors in RNAs synthesized in vitro. These mistakes, which include omission of encoded nucleotides as well as misinsertions, occur only on templates that support editing. The pattern of these misediting events indicates that editing site recognition and nucleotide addition are separable events, and that the recognition step involves features of the mitochondrial template that are required for editing. The larger deletions lack all templated nucleotides between editing sites, suggesting that the transcription/editing apparatus can ‘jump’ from one insertion site to another, perhaps mediated by interactions with editing determinants, while smaller omissions most likely reflect misalignment of the transcript upon resumption of templated RNA synthesis.

RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.

The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.

Publisher Summary This chapter describes the biochemistry of the interaction of phage coat protein with RNA and attempt to provide a molecular understanding of its high specificity. Coat protein binding is believed to serve two functions in the life cycle of the phage: 1) it acts as a translational repressor of the replicase gene early in infection, and 2) as an initiation site of phage assembly late in infection. This interaction has been extensively a prototype of sequence specific RNA-protein interactions. It is now clear that the phage coat proteins can be considered an example of a class of RNA hairpin binding proteins that are quite common in prokaryotes and eukaryotes. In case of bacteriophage coat protein, the coat protein assembles into phage-like capsids that can be purified by differential centrifugation and ion-exchange chromatography. Most coat proteins can be successfully renatured by the transfer from storage buffer directly into a variety of neutral buffers of moderate ionic strength. In many cases, these renatured proteins are fully active in both RNA binding and capsid assembly.

RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions. One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe. These mapped in or near the unlinked genes nrdB and nrdC. A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro. Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4.

Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the three introns. All three introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.

The td, nrdB, and sunY introns of bacteriophage T4 each contain a long open reading frame (ORF). These ORFs are preceded by functional T4 late promoters and, in the case of the nrdB intron ORF, a functional middle promoter. Expression of phage-encoded intron ORF-lacZ fusions indicates that these T4 genes are highly regulated. The lack of translation of these ORFs from early pre-mRNAs can be accounted for by the presence of secondary structures that are absent from the late RNAs. Because translation of the intron ORFs could disrupt core structural elements required for pre-mRNA splicing, such regulation may be necessary to allow expression of the genes in which they reside.