Insertion Editing Research Articles

RNA sequence and base pairing effects on insertion editing in Trypanosoma brucei.

RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.

Endoribonuclease activities of Trypanosoma brucei mitochondria

RNA editing in kinetoplastids is a type of post-transcriptional processing that changes mitochondrial mRNA sequences by the addition or deletion of uridines. Multiple enzymatic activities, such as endoribonuclease and RNA ligase, are associated with this process and exist in a multienzyme complex. Endonuclease activities from Trypanosoma brucei mitochondrial extracts were fractionated by sequential ion exchange and gel filtration chromatography. The RNA editing specific endonuclease activity co-fractionated with in vitro editing while another endonuclease activity with a different substrate specificity, and the majority of mtRNase P activity fractionated away from the editing activity. The pH, salt, temperature, and Mg 2+ optima of all three endonucleases were determined. All three activities are sensitive to high temperature and protease digestion. In addition, treatment with micrococcal nuclease resulted in partial disruption of the editing complex and decreased pre-cleaved in vitro insertion editing activity, suggesting that both RNA(s) and protein(s) are necessary in the intact functional complex.

Insertional editing of mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes.

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G. C base pair. Uridine insertions have been identified in the T loop of tRNALys from Didymium and tRNAGlu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTPsiC (Psi = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.

Insertional editing of nascent mitochondrial RNAs in Physarum.

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.

Defining the editing ‘reaction’

E xactly a decade after the initial report of RNA editing in the mitochondrion of the parasitic protozoan Trypanosoma brucei by Benne and co-workers l, the mechanism of uridylate (U) insertion and deletion has been resolved. In a recent article published in Science 2, Moffett Kable, Scott Seiwart and Stefan Heidmann, all working in Ken Stuart's laboratory, were able to establish, in vitro, an accurate U insertion RNA-editing reaction. The 'reaction' proceeds via a series of enzyme-catalyzed steps, with both the site and number of inserted Us directed by small transacting RNAs. This report follows closely on the heels of recent studies reporting the mechanism of deletion editing 3'4. These initial reports have been verified by others s. The papers from the Stuart lab have answered many of the fundamental questions concerning the general mechanism of RNA editing in trypanosomes. An inventory of the mitochondrial mRNAs of T. brucei reveals that 12 out of 17 mRNAs are processed by U insertion or deletion 6,v. The cytochrome oxidase II mRNA is only modestly changed, by the addition of four Us within the 663nucleotide transcript 1, from the sequence encoded by the mitochondrial genome. In contrast, in cytochrome oxidase III mRNA, the extent of editing is bewildering with 547 Us added and 41 Us deleted, resuiting in an mRNA that is so radically changed from the gene that greater than half of its residues are formed by RNA editing 8. In order to achieve the precision necessary to edit an mRNA correctly with this number of insertions and deletions, a template was postulated 9. A group of small, heterogeneous RNAs was identified as a candidate for directing the editing of mRNA. These guide RNAs (gRNA) are largely encoded by the minicircle DNA in the mitochondrion of trypanosomes and are complementary to short segments of edited mRNA. The gRNAs were also found in mitochondrial ribonucleoprotein complexes with pre-edited mRNAs and several enzymatic activities postulated to be involved in RNA editingl°'lL However, direct evidence of the role of gRNAs in RNA editing awaited the development of an in vitro system for accurate deletion and insertion editing. The recent studies from the Stuart lab have provided such a system. Their studies indicate that gRNAs have a dual role in editing. First, gRNAs recognize editing sites within the pre-edited mRNA and form the initial editing complex. This is mediated by the formation of a short 7-10-bp duplex between the gRNA and mRNA sequences immediately 3' to the editing site. This duplex is required for the subsequent enzymatic steps in editing. Second, the gRNA directs the number of Us added or deleted at an editing site. The results presented in the recent Science papers show that the Us are added or deleted to form a complementary sequence with the gRNA. The results of Kable et al. 2 and Seiwert et al. 3,4 also provide a great deal of insight into the mechanism of RNA editing in trypanosomes. Two general mechanisms have been proposed. The first is an enzyme cascade model 12 in which the pre-edited mRNA is cleaved sequentially by an editing-site-specific endoribonuclease 13, Us are added to or deleted from the 3' end of the premRNA 5' cleavage product by the action of a terminal-U transferase (TUTase) TM and the two halves are rejoined by the action of a mitochondrial RNA ligase 1-5,16 to form the edited mRNA. The second model is very different and proposes that

Editing of the mitochondrial small subunit rRNA in Physarum polycephalum.

Post-transcriptional insertion, substitution or deletion of nucleotides in RNA (RNA editing) has been observed in RNAs from a number of organisms but always in messenger RNA or transfer RNA. We report here that the 17S rRNA of the mitochondrial ribosome of Physarum polycephalum is edited at 40 sites with single cytidine insertions. The locations of the editing sites are fairly evenly distributed throughout the RNA and do not correspond to any obvious feature of the primary sequence or secondary structure. In addition to these cytidine editing sites are editing sites in which a nucleotide other than cytidine is inserted. At two sites a uridine is inserted and at two sites adenosine residues are inserted. This is the first report of mixed nucleotide insertional editing. These results imply that the editing mechanism in Physarum may be different from those proposed for the kinetoplastid protozoa.

RNA editing by cytidine insertion in mitochondria of Physarum polycephalum

A corollary of the central dogma of molecular biology is that genetic information passes from DNA to RNA by the continuous synthesis of RNA on a DNA template. The demonstration of RNA editing (the specific insertion, deletion or substitution of residues in RNA to create an RNA with a sequence different from its own template) raised the possibility that in some cases not all of the genetic information for a trait residues in the DNA template. Two different types of RNA editing have been identified in mitochondria: insertional editing represented by the extensive insertion (and occasional deletion) of uridine residues in mitochondrial RNAs of the kinetoplastid protozoa and the substitutional editing represented by the cytidine to uridine substitutions in some plant mitochondria. These editing types have not been shown to be present in the same organism and may have very different mechanisms. RNA editing of both types has been observed in nonmitochondrial systems but is not as extensive and may involve still different mechanisms. Here we report the discovery of extensive insertional RNA editing in mitochondria from an organism other than a kinetoplastid protozoan. The mitochondrial RNA apparently encoding the alpha subunit of ATP synthetase in the acellular slime mould, Physarum polycephalum, is edited at 54 sites by cytidine insertion.

Implementation of Graphical Layout Editor for Geologic Applications: ABSTRACT

The increasing availability and sophistication of data processing technology have given geologists new insights into the interpretation and evaluation of geological data. In many instances, however, software and hardware limitations have prevented geologists from effectively combining the graphical results from many of these specialized packaged procedures. At Aramco, the emphasis has been toward providing geologists and geological technicians with a convenient, user-friendly approach to effectively merging the graphical results of the various software packages. Initially designed for an IBM graphics workstation, the graphics layout editor (GLE) offers the user a method of quickly merging or compositing the graphical results of the most frequently used geological software packages in a menu-driven, interactive environment. Applications of GLE technology not only allow the user to produce expanded or enhanced variations on the original graphical output, it also gives geologists the flexibility to conveniently experiment with combinations of graphical results which would otherwise be cost prohibitive owing to drafting complexities. To support final design and presentation, GLE in an interactive mode also provides high-quality text capability, allowing the cartographer to quickly build and annotate presentation quality composites. GLE techniques of graphical overlay, insertion, and interactive editing provide geologists with an infinite series of perspectives into geologic problem solving. End_of_Article - Last_Page 465------------