RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and Ϸ20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).any mitochondrial mRNAs in trypanosomatids are edited by the insertion and deletion of uridine nucleotides (Us) as directed by guide RNAs (gRNAs) (1). The key steps of RNA editing are the coordinated endonucleolytic cleavage of preedited mRNA (pre-mRNA); terminal uridylyl transferase or U-specific exoribonuclease-mediated insertion or deletion of Us, respectively; and ligation. These enzymatic activities are in an Ϸ20S multiprotein complex, the editosome, which contains at least 20 proteins (2). The proteins responsible for U insertion (KRET2), deletion (KREX1), and ligation (KREL1 and KREL2) have been identified (3-7), but the protein (or proteins) responsible for endonucleolytic cleavage remain unidentified.The RNA editing endonuclease activity by editosomecontaining extracts cleaves synthetic ATPase subunit 6 (A6) or cytochrome b (CYb) pre-mRNAs in vitro in a gRNA-directed manner (8-10). The cleavage occurs 5Ј to the pre-mRNA:gRNA anchor duplex, leaving a 3Ј hydroxyl and 5Ј phosphate at the cleavage site. Cleavage of insertion sites is inhibited by adenosine nucleotides, whereas cleavage of deletion sites is stimulated by these nucleotides (11), suggesting that there may be distinct endonucleases. Several editosome proteins identified by mass spectrometry have motifs suggestive of nuclease function and may be RNA editing endonucleases (2).Kinetoplastid RNA editing protein (KREP) B1, KREPB2, and KREPB3 have RNase III, U1-like Zn 2ϩ finger, and dsRNAbinding motifs indicative of endonucleases. RNase III and dsRNA-binding motifs are typical in bacterial and eukaryotic endonucleases, and U1-like Zn 2ϩ finger motifs imply RNA and protein interactions in a complex (12). The RNase III motifs of KREPB1, KREPB2, and KREPB3 all conserve the amino acids that are critical to endonuclease function (13). A report indicating that KREPA3 ...

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent. RationaleThe genetic divergence of species is affected by both DNA metabolic processes and natural selection. Processes contributing to genetic variation that are undetectable with intraspecific data may be detectable by inter-specific analyses because of the accumulation of signal over evolutionary time scales. As a consequence of the greater statistical power, there is interest in applying comparative analyses to address an increasing number and diversity of problems, in particular analyses that integrate sequence and phenotype. Significant barriers that hinder the extension of comparative analyses to exploit genome indexed phenotypic data include the narrow focus of most analytical tools, and the diverse array of data sources, formats, and tools available. Theoretically coherent integrative analyses can be conducted by combining probabilistic models of different aspects of genotype. Probabilistic models of sequence change underlie many core bioinformatics tasks, including similarity search, sequence alignment, phylogenetic inference, and ancestral state reconstruction. Probabilistic models allow usage of likelihood inference, a powerful approach from statistics, to establish the significance of differences in support of competing hypotheses. Linking different analyses through a shared and explicit probabilistic model of sequence change is thus extremely valuable, and provides a basis for generalizing analyses to more complex models of evolution (for example, to incorporate dependence between sites). Numerous studies have established how biological factors representing metabolic or selective influences can be represented in substitution models as specific parameters that affect rates of interchange between sequence motifs or the spatial occurrence of such rates [1][2][3][4]. Given this solid grounding, it is desirable to have a toolkit that allows flexible parameterization of probabilistic models and interchange of appropriate modules.There are many existing software packages that can manipulate biological sequences and structures, but few allow specification of both truly novel statistical models and detailed workflow control for genome scale datasets. Traditional phylogenetic analysis applications [5,6] typically provide a number of explicitly defined statistical models that are difficult to modify. One exception in which the parameterization of entirely novel substitution models was poss...

Trypanosoma brucei has three distinct ϳ20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ϳ20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.RNA editing recodes most of the mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridine nucleotides (U's) using information provided by guide RNAs (gRNAs) (58). RNA editing is developmentally regulated and is required for parasites that cycle between insect vector and mammalian host (54). Proteins that perform catalytic steps of RNA editing have been identified: endonucleases KREN1 or KREN2 cleave at deletion or insertion sites, respectively; terminal uridylyl transferase (TUTase) KRET2 adds U's at insertion sites; U specific exoribonuclease (exoUase) KREX1 removes U's at deletion sites; and ligases KREL1 or KREL2 rejoin mRNA fragments after U addition or removal (5,13,25,30,34,55,61). These catalytic steps are coordinated by the multiprotein editosomes that sediment at ϳ20S on glycerol gradients (9, 43). KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.KREN1 and KREN2 were shown to be RNA editing endonucleases using both RNA interference (RNAi) and conditional knockout cell lines (5, 61). Both KREN1 and KREN2 are essential for the normal growth of PF and BF cells, and repression of their expression by either method led to dramatic growth defects or death, respectively. Such repression of KREN1 eliminated in vitro cleavage by ϳ20S editosomes of a deletion site in a synthetic substrate modeled on ATPase subunit 6 (A6) pre-mRNA but did not alter cleavage of an insertion site in a substrate derived from the same mRNA. These studies and other data have shown that KREN1 is an editing endonuclease with a preference for sites from which U's are deleted (29). Similarly, repression of KREN2 eliminated in vitro cleavage at insertion ed...