Intrinsic and regulated properties of minimally edited trypanosome mRNAs.

Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing.

The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein.

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite's single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T.

Characterization of a Trypanosoma brucei Alkb homolog capable of repairing alkylated DNA.

Trypanosoma brucei encodes a protein (denoted TbABH) that is homologous to AlkB of Escherichia coli and AlkB homolog (ABH) proteins in other organisms, raising the possibility that trypanosomes catalyze oxidative repair of alkylation-damaged DNA. TbABH was cloned and expressed in E. coli, and the recombinant protein was purified and characterized.

The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process.

Trypanosoma brucei brucei: thymine 7-hydroxylase-like proteins.

Two genes from Trypanosoma brucei brucei are predicted to encode Fe(II)- and alpha-ketoglutarate-dependent enzymes related to fungal thymine 7-hydroxylase. Transcription of the thymine hydroxylase-like genes is up-regulated in the bloodstream form of the parasite over the insect form, whereas Western blot analysis indicates more cross-reactive protein in the latter life stage. The genes were cloned, the proteins purified from Escherichia coli, and both proteins were shown to bind Fe(II) and alpha-ketoglutarate, confirming proper folding.

Complex interactions in the regulation of trypanosome mitochondrial gene expression.

Trypanosomes undergo extreme physiological changes to adapt to different environments as they cycle between hosts. Adaptation to the different environments has evolved an energy metabolism involving a mitochondrion with an unusual genome. Recently, Aphasizhev and colleagues have identified two new protein complexes, a mitochondrial polyadenylation complex and a guide RNA stabilization complex, that provide novel insights into the coordinated expression of the mitochondrial genome.

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure.

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins.

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues.

Identification of pentatricopeptide repeat proteins in Trypanosoma brucei.

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles.

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required.

An intragenic guide RNA location suggests a complex mechanism for mitochondrial gene expression in Trypanosoma brucei.

In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events.

A historical perspective on RNA editing: how the peculiar and bizarre became mainstream.

The known examples of RNA editing now encompass a variety of alterations of RNA primary sequence that arise from base modifications, nucleotide insertions or deletions, and nucleotide replacements. Hence, the definition of RNA editing has evolved as new systems have been described. This chapter presents a historical perspective on some of the pivotal discoveries that helped direct the current avenues of research in the field of RNA editing.