Kinetoplastid RNA editing involves a 3′ nucleotidyl phosphatase activity

Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3′ nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3′ nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3′ phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation

Lexis and grammar of mitochondrial RNA processing in Trypanosomes

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover

'Gestalt,' Composition and Function of the Trypanosoma brucei Editosome.

RNA editing describes a chemically diverse set of biomolecular reactions in which the nucleotide sequence of RNA molecules is altered. Editing reactions have been identified in many organisms and frequently contribute to the maturation of organellar transcripts. A special editing reaction has evolved within the mitochondria of the kinetoplastid protozoa. The process is characterized by the insertion and deletion of uridine nucleotides into otherwise nontranslatable messenger RNAs. Kinetoplastid RNA editing involves an exclusive class of small, noncoding RNAs known as guide RNAs. Furthermore, a unique molecular machinery, the editosome, catalyzes the process. Editosomes are megadalton multienzyme assemblies that provide a catalytic surface for the individual steps of the reaction cycle. Here I review the current mechanistic understanding and molecular inventory of kinetoplastid RNA editing and the editosome machinery. Special emphasis is placed on the molecular morphology of the editing complex in order to correlate structural features with functional characteristics

Parasite-specific aptamers as biosynthetic reagents and potential pharmaceuticals.

Aptamers are short, synthetic nucleic acid molecules. They are generated by a Darwinian-type in vitro evolution method known as 'systematic evolution of ligands by exponential enrichment' (SELEX). SELEX represents an experimental platform to identify rare ligands with predetermined functionality from combinatorial nucleic acid libraries. Since its discovery about 20 years ago the method has been instrumental in identifying a large number of aptamers that recognize targets of very different chemistry and molecular complexity. Although aptamers have been converted into sophisticated biomolecular tools for a diverse set of technologies, only a limited number of aptamers have been selected as binding reagents for parasites or parasite-derived molecules. Here the published examples of aptamers that target Leishmania-, Trypanosoma- and Plasmodia-specific molecules are reviewed

Does 5S RNA from E. coli have a pseudoknotted structure?

Chemical modification and limited enzymatic hydrolysis on isolated E. coli 5S RNA have provided informations on the secondary- and tertiary structure compatible with pseudoknotted structures for the A- and B-conformers of the molecule. Changes in the accessibility and reactivity of nucleotides in loop C and at the stem of helix IV in two different 5S RNA conformers are highly suggestive for interactions between bases C35 to C37 with G105 to G107 for the A-form and C38 to U40 and A94 to G96 with additional interactions of C35, C37 with G98 and G100 for the B-form. In both cases the molecules are folded forming pseudoknots and two quasi--continuous double stranded helices with coaxial stacking. The two structures are in perfect agreement with the biochemical data concerning the stability of the molecule and the chemical reactivities of individual nucleotides of the 5S RNA A- and B-conformers

Mechanism of the gBP21-mediated RNA/RNA annealing reaction: matchmaking and charge reduction

The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. The protein co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests that gBP21 contributes its annealing activity to the RNA editing machinery. In support of this view, gBP21 was found to accelerate the hybridization of cognate guide (g)RNA/pre-edited mRNA pairs. Here we analyze the mechanism of the gBP21-mediated RNA annealing reaction. Three possible modes of action are considered: chaperone function, matchmaker function and product stabilization. We conclude that gBP21 works as a matchmaker by binding to gRNAs as one of the two RNA annealing reactants. Three lines of evidence substantiate this model. First, gBP21 and gRNAs form a thermodynamically and kinetically stable complex in a 1 + 1 stoichiometry. Secondly, gRNA-bound gBP21 stabilizes single-stranded RNA, which can be considered the transition state in the annealing reaction. Thirdly, gBP21 has a low affinity for double-stranded RNAs, suggesting the release of the annealed reaction product after the hybridization step. In the process, up to six ionic bonds are formed between gBP21 and a gRNA, which decreases the net negative charge of the RNA. As a consequence, the electrostatic repulsion between the two annealing reactants is reduced favoring the hybridization reaction

Trypanosoma brucei mitochondria contain RNA helicase activity.

Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites.

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper