Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

The secondary structure of guide RNA molecules from Trypanosoma brucei.

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.

Quantitation of RNA editing substrates, products and potential intermediates: implications for developmental regulation.

Kinetoplast mitochondrial RNA editing is the developmentally regulated post-transcriptional process of uridine insertion and deletion in mRNAs directed by short guide RNAs (gRNAs), which creates functional mRNAs. Two mechanisms are proposed: transesterification which predicts gRNA/mRNA chimeric intermediates, and enzymatic steps which allow but do not require chimeric intermediates. We quantitated the copy number of apocytochrome b (CYb) gRNAs, edited/unedited mRNAs and gRNA/mRNA chimeras in bloodstream and procyclic form cells of Trypanosoma brucei. Both forms have 35 copies/cell of two gRNAs. Bloodstream forms contain 15 unedited and edited CYb mRNA molecules/cell while procyclic forms have four times as much unedited and over 10 times as much edited mRNA. Chimera levels are very low, 350-5000-fold lower than unedited mRNA or gRNAs, but are over 10 times more abundant in procyclic than bloodstream forms. These results are consistent with chimeras being editing intermediates if their resolution is rapid in respect to their formation, although they could be non-productive byproducts of the editing reaction. Bloodstream chimera sequences differ from procyclic chimeras. These results indicate that developmental regulation is not by gRNA abundance and suggest that it occurs at the level of gRNA utilization possibly by changing abundance of unedited CYb mRNA.

Multiple guide RNAs for identical editing of Trypanosoma brucei apocytochrome b mRNA have an unusual minicircle location and are developmentally regulated.

We identified four different guide RNAs (gRNAs) that specify identical editing of Trypanosoma brucei apocytochrome b (CYb) mRNA, which indicates gRNA redundancy in T. brucei. All four gRNAs appear functional since they occur in chimeras, some of which contain an interesting gRNA 3' "extension." The gRNAs are encoded in different minicircles, rather than maxicircles as in other species. However, these gRNA genes are not between 18-base pair repeats as are the other minicircle gRNA genes in T. brucei. The three minicircles cloned contain the same gRNA genes, one of which is substantially diverged, all in the same order, indicating that they are related. CYb gRNA is less abundant in procyclic than bloodstream forms. Procyclic forms contain abundant edited CYb mRNA unlike bloodstream forms thus suggesting that CYb mRNA editing may be regulated at the level of gRNA utilization.

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans.

The prestalk and prespore cells from the Dictyostelium discoideum multicellular slug stage of development differ in assembly of glycoconjugates. Prespore cells are 2- to 3-fold more active than prestalk cells in the assembly of N-linked glycans and 20-fold more active in their fucosylation. Only prespore cells synthesize an O-linked glycan consisting in part of Fuc alpha-linked to N-acetylglucosamine. Incorporation of fucose, glucosamine, mannose and galactose into large pronase-resistant glycoconjugates was almost exclusively into prespore cells. Such glucosamine-labelled glycoconjugates resist fragmentation by beta-elimination and include a glycoantigen dependent on the modB genetic locus. In contrast, large fucose-labelled glycoconjugates consisted of multiple, small, O-linked oligosaccharides on carrier peptides. The spore coat protein SP96 has several fucosylated O-linked oligosaccharides, one of which correlates with a fucose epitope previously shown to localize in prespore vesicles and the outer layer of the spore coat.

Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei.

RNA editing of several mitochondrial transcripts in Trypanosoma brucei is developmentally regulated. The cytochrome b and cytochrome oxidase II mRNAs are edited in procyclic-form parasites but are primarily unedited in bloodstream forms. The latter forms lack the mitochondrial respiratory system present in procyclic forms. Editing of the NADH dehydrogenase 7 (ND7) and ND8 transcripts is also developmentally regulated but occurs preferentially in bloodstream forms. Other transcripts, cytochrome oxidase III and ATPase 6, are edited in both life forms. We have identified many minicircle-encoded guide RNAs (gRNAs) for ATPase 6, ND7, and ND8. The characteristics of these gRNAs reveal how extensively edited RNA can be edited in the 3'-to-5' direction. Northern (RNA) blot and primer extension analyses indicate that gRNAs for transcripts whose editing is developmentally regulated are present in both procyclic and bloodstream form parasites. These results suggest that the developmental regulation of editing in these transcripts is not controlled by the presence or absence of gRNAs.

Positive and negative control of the Antennapedia promoter P2.

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. The maternally-active gene osk is identified as one of two general repressors of P2 which prevent Antp transcription until division cycle 14. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni then act as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp.

Differential regulation of glycoprotein sulfation and fucosylation during growth of Dictyostelium discoideum.

During early starvation-induced development, amoebae of Dictyostelium discoideum have been previously shown to increase sulfation and fucosylation of glycoprotein-linked oligosaccharides to levels above those observed in axenically growing cells. We report here that the axenic broth culture itself induces generation of high levels of fucosylated glycoprotein-linked oligosaccharides at all stages in the growth curve. However, when grown on bacteria, amoebae of both the axenic strain and the wild type show dramatic depression in fucose incorporation during early exponential growth. In mid- and late-exponential stages of growth, fucosylation rises to the levels found at all stages of axenic culture. Sulfation also increases during early development, but, in contrast to fucosylation, oligosaccharide sulfation is not altered by growth in axenic medium and does not increase during growth on bacteria. Starvation of bacterially grown cells results in increased sulfation and a further rise in fucosylation, as is also characteristic of broth-grown cells. The ability of axenic culture to uncouple control of these two classes of glycan-modification steps suggests that the synchronous increases during early development actually reflect responses to different regulatory signals, even though they participate in the same metabolic process. The increase in in vivo fucosyltransferase activity, which can act on many substrate glycoproteins, may alter many characteristics of the cells.