Biomedical subjects

Sean F O'Hearn

Publications and source records attributed to Sean F O'Hearn.

5 recordsLinked to original sources

PubMed2006

T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion cycle.

Trypanosome RNA editing is massive post-transcriptional U-insertion and U-deletion, which generates mature mRNA coding regions through cycles of endonuclease, terminal U transferase (TUTase) or 3'-U-exo, and ligase action. Both types of editing are thought to be catalyzed by distinct sets of proteins of a multiprotein complex, and no enzymatic activity of wild-type editing complex had been shown to function in both forms of editing. By examining the individual steps of the U-deletion cycle using purified editing complex, traditional mitochondrial extract, and rapidly prepared cell lysate, we here demonstrate that TbMP57 TUTase of U-insertion can act efficiently within a U-deletion cycle. When physiological UTP levels are provided, it adds U's to the upstream cleavage fragment after U-deletional endonuclease and 3'-U-exo action, but before rejoining by the U-deletional ligase, generating partial U-deletion products. TUTase activity in U-deletion was not previously appreciated since its detection requires UTP, which is not normally added to in vitro U-deletion reactions. Fractionation and RNAi analyses show this U-addition in U-deletion requires TbMP57 TUTase be present and competent for U-insertion; such U-addition does not occur with another mitochondrial TUTase that is separate from the basic editing complex. Efficient TbMP57 action in both U-insertion and U-deletion suggests these two editing forms may be less separate than generally envisioned. Should such promiscuous TUTase action also occur in vivo, it could explain why editing utilizes substantially fewer U-deletional than U-insertional events and why partial editing appears preferential in U-deletion.

Animals↗

PubMed2005

Molecular research technologies in mitochondrial diseases: the microarray approach.

Mitochondria are ubiquitous in eukaryotic cells where they generate much of the cellular energy by the process of oxidative phosphorylation (OXPHOS). The approximately 1500 genes of the mitochondrial genome are distributed between the cytoplasmic, maternally-inherited, mitochondrial DNA (mtDNA) which encodes 37 genes and the nuclear DNA (nDNA) which encompasses the remaining mitochondrial genes. The interplay between the mtDNA and nDNA encoded mitochondrial genes and their role in mitochondrial disorders is still largely unclear. One approach for elucidating the pathophysiology of mitochondrial diseases has been to look at changes in the expression of mtDNA and nDNA-encoded genes in response to specific mitochondrial genetic defects. Initial studies of gene expression changes in response to mtDNA defect employed blot technologies to analyze changes in the expression of individual genes one at a time. While Southern/Northern blot experiments confirmed the importance of nDNA-mtDNA interactions in the pathophysiology of mitochondrial myopathy, the methodology used limited the number of genes that could be analyzed from each patient. This barrier has been overcome, in part by the advent of DNA microarray technology. In DNA microarrays gene sequences or oligonucleotides homologous to gene sequences are arrayed on a solid support. The RNA from the subject is then isolated, the mRNA converted to cDNA and the cDNA labeled with a fluorescent probe. The labeled cDNA is hybridized on the microarray and the fluorescence bound to each array is then quantified. Recently, these technologies have been applied to mitochondrial disease patient tissues and the presence of coordinate changes in mitochondrial gene expression confirmed.

DNA, Mitochondrial↗

PubMed2005

In Trypanosoma brucei RNA editing, band II enables recognition specifically at each step of the U insertion cycle.

Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

Animals↗

PubMed2003

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

Amino Acid Sequence↗

PubMed2002

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3'-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Amino Acid Sequence↗