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Kenneth Stuart

Publications and source records attributed to Kenneth Stuart.

29 records · Page 2Linked to original sources
PubMed2003

TbMP44 is essential for RNA editing and structural integrity of the editosome in Trypanosoma brucei.

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.

Alleles↗
PubMed2003

Identification of novel components of Trypanosoma brucei editosomes.

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed.

Amino Acid Sequence↗
PubMed2002

TbMP81 is required for RNA editing in Trypanosoma brucei.

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.

Animals↗
PubMed2002

A low-background inducible promoter system in Leishmania donovani.

We report here a second-generation tetracycline-responsive repressor-operator system in Leishmania donovani. In this system, expression of a reporter luciferase gene (LUC) is driven by the inducible Leishmania ribosomal RNA promoter on the DNA strand opposite to a hygromycin resistance gene (HYG) whose expression is driven by the endogenous pol I promoter on chromosome 27 (rDNA locus) or the endogenous pol II promoter on chromosome 35 (LD1 locus). Transgenic cell lines showed regulation of LUC gene expression over three orders of magnitude. In the absence of tetracycline, luciferase expression levels were 2-3-fold higher than machine background when integrated into the LD1 locus, but was over 10-fold higher than machine background when integrated into the rDNA locus.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

Animals↗
PubMed2002

RNA editing: complexity and complications.

RNA editing in Trypanosomatids creates functional mitochondrial mRNAs by extensive uridylate (U) insertion and deletion as specified by small guide RNAs (gRNAs). Editing is catalysed by the multiprotein editosome. Over 20 of its protein components have been identified and additional proteins are likely to function in editing and its regulation. The functions of only a few editosome proteins have been determined. Surprisingly, there are related pairs or sets of editosome proteins, and insertion and deletion editing appear to be functionally and perhaps spatially separate. A model for the editosome is proposed, which has a catalysis domain with separate sectors for insertion and deletion editing. It also contains domains for anchor duplex and upstream RNA binding, which position the sequence to be edited in the catalysis domain.

Animals↗
PubMed2002

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing.

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3' exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3' pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5' phosphate of the 3' fragment, which is not required for U removal. The ability of the 3' U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3' exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp)alpha-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

Animals↗
PubMed2002

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.

Animals↗
PubMedDate not supplied

Trypanosomatid flagellum biogenesis: ARL-3A is involved in several species.

Overexpression in Leishmania amazonensis promastigotes of the GTPase-deficient small G protein LdARL-3A-Q70L specifically provokes the loss of the flagella without affecting cell viability and body size. However, motility is lost and, remarkably, cells do not survive in the insect vector Lutzomyia longipalpis gut, leading to interruption of parasite transmission. We report here that overexpression of the same protein in Leishmania major, Leishmania donovani, and Crithidia fasciculata also led to significant alterations of the flagella. Surprisingly, ablation of TbARL-3A expression by RNAi in Trypanosoma brucei brucei also provoked flagella shortening, revealing that overexpression of the GTPase-deficient protein seems functionally equivalent to a drastic reduction in its native counterpart abundance. This renders possible complementary studies of an essential pathway in related organisms. Potential significance for the protein function is discussed as well as future strategies for stopping the transmission of several neglected parasitic diseases.

Amino Acid Sequence↗