Ph.D. / Molecular Biology / Neurospora crassa arg-2 gene encodes the small subunit of arginine specific carbamoyl phosphate synthethase, the first enzyme in fungal arginine (Arg) biosynthesis. The arg-2 mRNA contains an upstream open reading frame (uORF) specifying an evolutionarily conserved 24-residue peptide called the arginine attenuator peptide (AAP). Synthesis of the AAP causes ribosomes to stall on the mRNA in the presence of high concentrations of Arg. The amino acid sequence of the AAP, and not the sequence of its coding region, is responsible for this regulation. Scanning mutagenesis within the evolutionarily conserved region revealed that some residues are more important than others for the AAP to function. While most known nascent peptides that regulate translation are found encoded as uORFs or as N-terminal leader peptides, the AAP can exert regulatory function whether placed near the N-terminus or internally within a large polypeptide. The AAP’s peptide sensing features are conserved due to regulated stalling of fungal, plant, and animal ribosomes in response to Arg. That the AAP functions as an internal domain to regulate elongation in response to Arg establishes that such domains can provide a means of controlling translational elongation. The minimal sequences required for AAP to function as an internal domain was revealed by systematic deletion of its natural N- and C-terminal regions. Comparative analysis of the AAP with other fungi showed that the evolutionarily conserved region of the peptide is required for regulation. This minimal domain functions when placed as a uORF as seen by toeprint assay. Analyses of Arg analogs provided key insights in the structural requirements for Arg’s role in regulation. These results taken together provide a detailed picture of the requirements for Arg-specific regulation mediated by the AAP.

No description available.

Ribosomes are cellular nanomachines responsible for protein production in all living cells. When ribosome biogenesis is compromised, or ribosome function unfaithful, it causes diseases called ribosomopathies. The primary goal of my PhD was to understand the consequences of ribosome biogenesis dysfunction on translation. I have contributed to this understanding through four different projects which were aimed to understand how ribosome function affects the different steps of protein translation in the cell. In my first project, we tested if a ribosomal RNA sugar methylation present on the large ribosomal subunit plays a role in translation. We found that the loss of the modification does not grossly inhibit ribosome production or growth. However, these mutants are resistance towards G418, and make fewer decoding errors as compared to the control cells. In my second project, I studied a methyltransferase called Mtq2, which methylates the translation termination release factor eRF1. We found that Mtq2 is directly involved in late steps of large ribosomal subunit maturation and that the catalytic activity of Mtq2 is required for efficient 60S subunit production and for pre-60S export. In project 3, I studied a natural, plant-derived alkaloid called haemanthamine (HAE). We showed that HAE binds the peptidyl transferase center of the large subunit of the eukaryotic ribosome, where it interacts with the 25S rRNA. We also showed that HAE inhibit early stages of pre-rRNA processing and elicit nucleolar stress response in the cells. In project 4, I studied a long non-coding RNA called SAMMSON. SAMMSON plays a crucial role in melanoma survival. We found that depletion of SAMMSON adversely affects ribosome biogenesis. We also demonstrated that by modulating the binding affinity of a single protein, namely CARF, SAMMSON rewires the RNA-protein network and promotes a synchronized increase in rRNA maturation both in the cytosol and mitochondria, thereby boosting translation in both the cellular compartments. / Les ribosomes sont des nanomachines cellulaires responsables de la production de protéines dans toutes les cellules vivantes. Lorsque la biogenèse des ribosomes est compromise ou que la fonction des ribosomes est infidèle, elle provoque des maladies appelées ribosomopathies. L'objectif principal de ma thèse était de comprendre les conséquences du dysfonctionnement de la biogenèse des ribosomes sur la traduction. J'ai contribué à cette compréhension par le biais de quatre projets différents visant à comprendre comment la fonction des ribosomes affecte les différentes étapes de la traduction des protéines dans la cellule. Dans mon premier projet, nous avons voulu determiner si une méthylation sur l’ARN ribosomique d’un sucre présente sur la grande sous-unité ribosomique joue un rôle dans la traduction. Nous avons constaté que la perte de cette modification n'inhibait pas grossièrement la production ou la croissance des ribosomes. Cependant, ces mutants sont résistants à G418 et font moins d’erreurs de décodage par rapport aux cellules contrôles. Dans mon deuxième projet, j'ai étudié une méthyltransférase appelée Mtq2, qui méthyle le facteur de libération de la terminaison de la traduction, eRF1. Nous avons constaté que Mtq2 est directement impliqué dans les dernières étapes de la maturation des grandes sous-unités ribosomiques et que l'activité catalytique de Mtq2 est nécessaire pour une production efficace de sous-unités 60S et pour une exportation antérieure à 60S. Dans le cadre du projet 3, j'ai étudié un alcaloïde naturel d'origine végétale appelé hémanthamine (HAE). Nous avons montré que HAE lie le centre de la peptidyl transférase de la grande sous-unité du ribosome eucaryote, où il interagit avec l'ARNr 25S. Nous avons également montré que HAE inhibe les stades précoces du traitement pré-ARNr et induit une réponse au stress nucléolaire dans les cellules. Dans le projet 4, j'ai étudié un long ARN non codant appelé SAMMSON. SAMMSON joue un rôle crucial dans la survie du mélanome. Nous avons constaté que sa perte d’expression affecte négativement la biogenèse des ribosomes. Nous avons également démontré qu'en modulant l'affinité de liaison d'une protéine unique, à savoir CARF, SAMMSON réarme le réseau ARN-protéine et favorise une augmentation synchronisée de la maturation de l'ARNr à la fois dans le cytosol et les mitochondries, renforçant ainsi la traduction dans les deux compartiments cellulaires. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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No description available.

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Catherine B. Kirn-Safran, Dept. of Biological Sciences. Includes bibliographical references.

Ribosomes are multicomponent macromolecular particles and are essential for the survival of cells in all organisms. The ribosome's universal function is to catalyze polypeptide synthesis through translation of mRNA transcripts. Ribosomes from Escherichia coli, eubacterial organisms, have a sedimentation coefficient of 70S and are composed of 30S and 50S ribonucleoprotein subunits. The small ribosomal subunit is an assembly of 21 different proteins and a 16S ribosomal RNA. Within the 16S rRNA there are a few short stretches of universally conserved sequences spanning positions 517-533, 1394-1408, and 1492-1506. Clear functions for these sequence zones have not yet been assigned. Here I report a kinetic analysis of these highly conserved regions in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity was measured in experiments that were based on protection from nuclease 51 digestion of short oligodeoxynucleotides hybridized to the designated regions. DNAs hybridized to regions 1400 and 1500 show significant differences in the apparent dissociation constants when measured in 30S particles as opposed to those found for 16S rRNA. Region 525 showed no difference in kinetic behavior. To further elucidate the functional and structural role played by the region centered about C1400 in 16S rRNA, a four nucleotide deletion was constructed within this region. The deletion was introduced by direct RNA manipulation using DNA/RNA hybridization, RNase H digestions, and ligation of the correct RNA fragments with T4 RNA ligase. I improved ligation efficiency of large RNA molecules by including a connector looped short DNA oligomer. Recycling products through phenyl boronate agarose (PBA-30) column also improved the efficiency of ligation. The mutagenized 16S rRNA fully reassembles into 30 particles and the altered 30S subunit possesses all of the normal ribosomal proteins. Altered ribosomes were functional in in vitro translation of MS2 mRNA. The altered ribosomes have lower translational activity relative to controls. Here I present indirect evidence suggesting that the decrease in the synthesis of MS2 coat proteins is the result of premature termination. The altered 16S RNA in ribosomes had an apparent dissociation constants with DNA probes comparable to those found for normal 16S rRNA. This suggest that the RNA is less flexible in the particle relative to normal 30S subunits. The deletion at 1400 did not have any effect on the physical properties of the 1500 region, as measured by DNA hybridization. A minor, but significant, effect on the 525 region was observed. A possible RNA/RNA interaction within the 30S particle is proposed to account for this observation. / Graduation date: 1992

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 38-43).

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.