Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Akron, Dept. of Biology, 2006. / "December, 2006." Title from electronic thesis title page (viewed 12/31/2008) Advisor, Robert Joel Duff; Committee members, Richard Londraville, Francisco B. Moore, Amy Milsted; Department Chair, Bruce Cushing; Dean of the College, Ronald F. Levant; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

This thesis is a follow-up of my Bachelor thesis about the mitochondrial genome of kinetoplastid protist Perkinsela sp. This work introduces a novel approach in high-throughput analysis method of uridine insertion and deletion RNA editing, describes its background and proposes its further development. Its effect on the interpretation of U-indel editing, both in Perkinsela and in general, is demonstrated via attached manuscript which also introduces other biologically relevant aspects of Perkinsela mitochondrion.

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Il serait opportun de débuter cette introduction en donnant une définition claire du processus d'édition des ARN, mais c'est aussi un exercice périlleux car le terme d'édition des ARN a été utilisé dans la littérature pour décrire une multitude de processus biochimiques différents et la distinction entre les processus d'édition ou de modification est parfois confuse. Le terme d’édition des ARN a été utilisé pour la première fois en 1986 pour décrire l’insertion de 4 résidus uridines dans le transcrit mitochondrial coxII chez le trypanosome (Benne et al., 1986). La communauté scientifique était sceptique et on a alors pensé que ce mécanisme était sans doute spécifique à ce « drôle » de protozoaire. Puis, rapidement, l'édition d'ARNm a été décrite chez de nombreux organismes eucaryotes, soit pour expliquer des processus d'insertions ou de délétions de nucléotides (qui altèrent le nombre de nucléotides contenus dans la molécule d'ARN) soit pour décrire des conversions ou des remplacements de nucléotides (qui altèrent l'identité des nucléotides contenus dans la molécule d'ARN). Plus tard, le terme d'édition des ARN a été utilisé pour décrire des désaminations (le plus fréquemment C-en-U, et A-en-I) survenant dans les ARNt et les ARNr d'organismes eucaryotes et procaryotes, mais aussi des modifications mineures des résidus (comme l'ajout de groupement méthyl). De même la polyadénylation de la partie 3' de certains ARNt est aussi communément appelée processus d'édition des ARN. Enfin, un phénomène d'édition cotranscriptionnel des ARN lié au « patinage » de l'ARN polymérase a également été mis en évidence chez certains virus.

ADAR (Adenosine Deaminases acting on RNA) family proteins are double-strand RNA binding proteins that deaminate specific adenosines into inosines. This A-to-I conversion is called A-to-I RNA editing and is well conserved in the animal kingdom from nematodes to humans. RNA editing is a pre-splicing event on nascent RNA that may affect alternative splicing when the editing occurs in the exon-intron junction or in the intron. Also, editing may change biological function of small RNAs by editing the premicroRNAs or other noncoding RNAs. Editing also alters protein amino acid sequences because inosine in the mRNA base pairs with cytosine and is therefore read as guanosine. In mammals, there are three ADAR family proteins, ADAR1, ADAR2, and ADAR3, encoded by three different genes. So far, no enzymatic activity of ADAR3 is detected. The most frequently edited targets of ADAR1 and ADAR2 are regions covering copies of Alu transposable elements in primates. In addition, loss of some specific editing events leads to profound phenotypes when the editing does not occur correctly. For example, some human neural disorders – such as epilepsy, forebrain ischemia, and Amyotrophic Lateral Sclerosis – are known to be associated with abnormally edited ion channel transcripts. Drosophila has a single ADAR protein (encoded by the Adar gene) that is highly conserved with human ADAR2 (encoded by the ADARB1 gene). To date, 972 editing sites have been identified in 597 transcripts in Drosophila, and approximately 20% of AGO2-associated esiRNAs are edited. Similar to mammals, many ion channel-encoding mRNA transcripts undergo ADAR-mediated A-to-I editing in Drosophila. While Adar1 null mice die at the embryonic stage and Adar2 null mice die shortly after birth due to seizures, Adar null flies are morphologically normal and have normal life span under ideal conditions. However, Adar null flies exhibit severe neurodegeneration and locomotion defects from eclosion, whilst Adar overexpression (OE) is lethal. To better understand the physiological role of RNA editing and ADAR, and to shed light on ADAR-related human disease, I used Drosophila Adar mutant flies as a model organism to investigate phenotypes, and to find chromosomal deletions and specific mutations that rescue the neural-behavioural phenotype of the Adar null mutant flies. Using the publicly available chromosomal deletions collectively covering more than 80% of the euchromatic genome of Chromsome III, I performed a genetic screen to find rescuers of the lethality caused by Adar overexpression. I confirmed that mutation in Rdl (Resistant to dieldrin, the gene encoding GABAA receptor main subunit) rescues. This rescue was not likely caused by effects on Adar expression level or activity. Driven by the hypothesis that the rescue may be due to reduction in GABAergic input to neurons, I recorded spontaneous firing activity of Drosophila larval aCC motor neurons using in vivo extracellular current recording technique. As expected, the neurons overexpressing Adar had much less activities compared with wild type neurons. Also, I found that Adar null fly neurons fired much more and showed epilepsy-like increased excitability. Although feeding PTX (Picrotoxin), a GABAA receptor antagonist, failed to rescue the lethality, reducing the expression of GAD1 to reduce synthesis of GABA was able to rescue the ADAR overexpression lethality. These results suggest that ADAR may finetune neuron activity synergistically with the GABAergic inhibitory signal pathway. I used MARCM (mosaic analysis using a repressible cell marker) to detect cellautonomous phenotypes in Adar null cells in otherwise wild type flies. Although neurodegeneration, observed as enlarged vacuoles formation in neurophils, was detected both in histological staining and EM images, the Adar null neurons marked with GFP from early developmental stages were not lost with age. Nevertheless, swelling in the axons or fragmentation of the axon branches of Adar null neurons was sometimes observed in the midbrain. By comparing the Poly-A RNA sequencing data from Adar null and wild type fly heads, we detected significant upregulation of innate immune genes. I confirmed this by qRT PCR and found that inactive ADAR reduces the innate immune gene transcript levels almost as much as active ADAR does. Further, using the locomotion assay, I confirmed that reintroducing inactive ADAR into Adar null flies can improve the flies’ climbing ability. Based on the Adar null flies having comparatively low viability, I performed a second deficiency screen to find rescuers of Adar null low viability using the same set of deficiencies as in the lethality rescue screen described above. I found seven deletions removing 1 to 37 genes that significantly increased the relative viability of the Adar null flies. However, not all the rescuing deficiencies also improved the Adar null locomotion. One rescuing gene, CG11357 was mapped from one of the rescuing deficiencies, and some mutant alleles of cry, JIL-1 and Gem3 also showed significant effects on the Adar null fly viability. The single gene viability rescuers were also not necessarily locomotion or neurodegeneration rescuers. Although the initial aim was to find neural-behavioural rescuing genes from the viability screen, the viability rescuers found in the screen are more likely to play a role in different aspects of stress response for survival.

The mitochondrial genome (kinetoplast or kDNA) of Trypanosoma brucei is highly complex in terms of structure, content and function. It is composed of two types of molecules: 10-50 copies of identical ~23-kb maxicircles and 5,000-10,000 highly heterogeneous 1-kb minicircles. Maxicircles and minicircles form a concatenated network that resembles chainmail. Maxicircles are the equivalent of mitochondrial DNA in other eukaryotes, but 12 out of the 18 protein-coding genes encoded on the maxicircle require post-transcriptional RNA editing by uridylate insertion and removal before a functional mRNA can be generated. The 1-kb minicircles make up the bulk of the kDNA content and facilitate the editing of the maxicircle-encoded mRNAs by encoding short guide RNAs (gRNAs) that are complementary to blocks of edited sequence. It is estimated that there are at least hundred classes of minicircle, each class encoding a different set of gRNAs. At each cycle of cell division the contents of the kDNA genome must be faithfully copied and segregated into the daughter cells. Mathematical modelling of kDNA replication has shown that failure to segregate evenly will eventually result in loss of low copy number minicircle classes from the population. Depending on the type of minicircle that is lost this can result in immediate parasite death or, if the loss occurred in the bloodstream stage, render the cells unable to complete the canonical life-cycle in the tsetse fly vector. In order to investigate minicircle complexity and replication in T. brucei further we i) first established the true complexity of the kDNA genome using a Illumina short read sequencing and a bespoke assembly pipeline, ii) annotated the minicircles to establish the editing capacity of the cells, iii) analysed expression levels of predicted gRNA gene cassettes using small RNA data, and iv) carried out a long term time course to measure how kDNA complexity changes over time and compared this to preliminary model predictions. The structure of this thesis follows these steps. Using these approaches, 365 unique and complete minicircle sequences were assembled and annotated, representing 99% of the minicircle genome of the differentiation competent (i.e. transmission competent) T. brucei strain AnTat90.13. These minicircles encode 593 canonical gRNAs, defined as having a match in the known editing space, and a further 558 non-canonical gRNAs with unknown function. Transcriptome analysis showed that the non-canonical gRNAs, like the canonical set, have 3' U-tails and showed the same length distribution. Canonical and non-canonical sets differ, however, in their sense to anti-sense transcript ratios. In vitro culturing of bloodstream form T. brucei for ~500 generations resulted in loss of ~30 minicircle classes. After incorporating parameters for network size and minicircle diversity determined above, model fitting to longitudinal kDNA complexity data will provide estimations for the fidelity of kDNA segregation. The refined mathematical model for kDNA segregation will permit insight into time constraints for transmissibility during chronic infections due to progressive minicircle loss. It also has the potential to shed light on the selective pressures that may have led to the apparent co-evolution of the concatenated kDNA network structure and parasitism in kinetoplastids.