Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis

Non-protein-coding RNAs represent a large proportion of transcribed sequences in eukaryotes. These RNAs often function in large RNA–protein complexes, which are catalysts in various RNA-processing pathways. As RNA processing has become an increasingly important area of research, numerous non-messenger RNAs have been uncovered in all the model eukaryotic organisms. However, knowledge on RNA processing in deep-branching eukaryotes is still limited. This study focuses on the identification of non-protein-coding RNAs from the diplomonad parasite Giardia intestinalis, showing that a combined experimental and computational search strategy is a fast method of screening reduced or compact genomes. The analysis of our Giardia cDNA library has uncovered 31 novel candidates, including C/D-box and H/ACA box snoRNAs, as well as an unusual transcript of RNase P, and double-stranded RNAs. Subsequent computational analysis has revealed additional putative C/D-box snoRNAs. Our results will lead towards a future understanding of RNA metabolism in the deep-branching eukaryote Giardia, as more ncRNAs are characterized

DNA typing of the human small intestinal protozoan parasite Giardia lamblia

PhDAt present there is no satisfactory means of typing strains of Giardia lamblia which can explain the broad range of clinical symptoms seen in giardiasis or which can identify genotypes in epidemiological studies. This thesis attempts to address these problems by developing DNA based typing systems sensitive enough to be able to identify many different Giardia genotypes and which may be applied to the organisms found in clinical samples. Four different techniques were assessed for their ability to identify multiple polymorphic loci in the Giardia genome which may be used to genotype and identify isolates of Giardia and upon which the future development of PCR-typing protocols may be based. These techniques included RFLP analysis, random amplified polymorphic DNA (RAPD) analysis, M13 DNA fingerprinting and minisatellite DNA fingerprinting. Minisatellite DNA fingerprinting proved to be the most discriminatory, recognising many hypervariable loci within the Giardia genome which proved useful for in vitro studies on genotypic heterogeneity within Giardia isolates. This approach would require further development in order to be used on in vivo infections where it could directly assess the relationship between genotype and pathogenicity. Therefore the variable repeats recognised on Giardia fingerprints were sought by constructing and screening a Giardia genomic DNA cosmid library. Once cloned these repeats would form the basis of sensitive and specific PCR-based fingerprinting protocols ideal for typing large numbers of infections. The repeat sequences cloned in this way turned out to be Giardia variable surface protein genes with short, imperfect tandem repeats in their 3' flanking DNA. This work has important implications for the future development and use of fingerprinting techniques on Giardia and may be useful in the study of chromosome rearrangement in Giardia which is likely to be involved in surface antigen switching

Konzervovaný mechanismus cílení Tail-anchored proteinů u eukaryot

Membránové proteiny, které tvoří přibližně jednu čtvrtinu všech proteinů v buňce, jsou transportovány skrze hydrofilní prostředí cytosolu, než jsou integrovány do membrány organel, nebo do plasmatické membrány. Jedná se o složitý proces, který vyžaduje správné načasování i souhru řady zúčastněných proteinů. Skupina transmembránových proteinů, tzv. Tail-anchored (TA) proteinů, neobsahuje klasický signální peptid umístěný na N-konci proteinu, který proteiny cílí k membráně endoplasmatického retikula. Tato informace je místo toho obsažena v jediné transmembránové doméně na C-konci TA proteinů. Z tohoto důvodu musí být TA proteiny transportovány až po ukončení translace. Hlavní způsob transportu TA proteinů je zprostředkován tzv. Guided Entry of Tail-anchored protein drahou neboli GET drahou. Tuto dráhu tvoří u kvasinek celkem šest (Sgt2, Get1-Get5) a u člověka sedm (navíc Bag6) proteinů, které mají za úkol rozeznat a navázat TA protein, přenést ho k membráně endoplazmatického retikula a zprostředkovat jeho vložení do membrány. Kromě modelových systémů, byly některé z proteinů GET dráhy studovány u rostlin a u Plasmodium falciparum. To do značné míry komplikuje naše pochopení obecného mechanismu dráhy i její využití napříč eukaryoty. Rozhodli jsem se proto charakterizovat GET dráhu u parazitického...Approximately one-fourth of all cellular proteins represent integral membrane proteins (IMPs) that are transported through the cytosol across or into the organellar or plasma membrane. Transport of IMPs requires precise timing which needs to be precisely regulated for them to reach their final destination. Tail-anchored (TA) proteins represent specific class of membrane proteins that lack the N-terminal signal peptide, which targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for the co-translational transport. Instead, they possess single C-terminal transmembrane domain (TMD) that serves as their targeting signal. Therefore, TA proteins are transported only post-translationally when the C-terminal TMD appears from the ribosome. The Guided Entry of Tail-anchored proteins (GET) pathway is the dominant way of how TA proteins find their way into the ER membrane. It is a multistep process that is mediated by six (Sgt2, Get1-Get5) proteins in yeast and seven (plus Bag6) proteins in human, which involves recognition of a TA protein, its targeting to the ER membrane and the actual membrane insertion. In addition to the model cell systems, some of GET pathway components were studied in plants and recently in Plasmodium falciparum, which makes our knowledge on the distribution and the...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Identification and comparison of non-coding RNAs and ribonucleoprotein complexes in several diverse protist species

Many different classes of non-coding (nc)RNAs are found in species throughout the tree of life, each of which performs important cellular functions. The objective of my thesis was to identify and characterize ncRNAs and their associated protein complexes, with particular focus on small nucleolar RNAs (snoRNA), in unicellular eukaryotes whose genomes have undergone significant reduction or expansion. To do this I utilized the unicellular eukaryotes Giardia lamblia, Giardia muris and Euglena gracilis. A diRNP containing RNase P and snoRNA domains that targets tRNAMet 2ʹ-O-methylation and the U3 snoRNA were discovered and characterized in two Giardia species. Unique binding/assembly properties were determined for C/D snoRNP proteins in G. lamblia. A large collection of E. gracilis snoRNAs were discovered leading to a better understanding Ψ-guide snoRNA structure and evolution. These findings highlight the diversity of ncRNA features that exist in less well studied eukaryotic species, and their unique functions

Systematic computational analysis of potential RNA interference regulation in Toxoplasma gondii

Thesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2009Includes bibliographical references (leaves: 58-73)Text in English; Abstract: Turkish and Englishx, 79 leavesRNA-mediated silencing was first described in plants and became famous by studies in Caenorhabditis elegans. RNA interference (RNAi) is the mechanism through which an RNA interferes with the production of other RNAs in a sequence specific manner. MiRNAs are a type of RNA which originate from the genome with their active form being ss-RNAs of 21-23 nucleotides in length. They are being transcribed as primiRNAs then processed in the nucleus by Drosha to pre-miRNAs with a stem-loop structure and 70 nucleotides in length. This stem-loop containing pre-miRNAs is then processed in the cytoplasm to ds-RNA one strand of which will serve as interfering RNA. Toxoplasma gondii is a species of parasitic protozoa which causes several diseases. T.gondii emerges as a good candidate for computational efforts with its small genome size, publicly available genome files and extensive information about its gene structure, either based on experimental data or the prediction with several gene finders in parallel. Therefore, it seems important to establish the regulatory network composed of RNAi which may be beneficial for the Toxoplasma community. Within this context the pool of possible stem-loop constitutive transcripts are produced, further analysis of this pool for desired 2D structure is integrated and mapping of possible RNAi regulation to T.gondii.s genome is established. In connection with computational assessment and mapping, the derived information is provided as a database for quick lookup using a convenient web interface for experimental studies of RNAi regulation in Toxoplasma, thus reduce time and money costs in such studies