Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Eugene G. Mueller, Dept. of Chemistry and Biochemistry. Includes bibliographical references.

RNA contains over 100 post-transcriptional chemical modifications. Of these, the most abundant is pseudouridine (Ψ), the 5’ ribosyl isomer of uridine. The formation of Ψ occurs at the polynucleotide level, and is catalyzed by Ψ synthase enzymes. To date, five families of Ψ synthases have been identified. Our work deals with the fifth and last family to be discovered, the Ψ synthase TruD family. TruD homologs are present in organisms across the three kingdoms of life. A sequence alignment of TruD homologs reveals distinct sequence insertions at specific and conserved locations in homologs from higher organisms. We have carried out extensive bioinformatics searches in an effort to characterize these insertion sequences, and have found that one of these insertions has a high degree of similarity to the R3H single-strand nucleic acid binding domain. To further understand the nature of this insertion, we examined its role in the enzymatic activity of the yeast TruD homolog, Pus7p, and found that mutating this insert decreased the enzyme’s activity by almost half. The human genome codes for two TruD homologs, PUS7 and PUS7L. These putative pseudouridine synthases were named based on their similarity to the yeast TruD homolog Pus7p, but their function has only been inferred based on sequence, and neither their activity, nor their structure have been examined. In an effort to further our understanding about the TruD synthase family, we have taken a closer look at PUS7 and PUS7L. We have determined the optimal conditions for over-expression of the enzymes in a bacterial expression system, and shown that the proteins are soluble, and can be purified using serial chromatography. In addition, we have queried these enzymes’ ability to recognize canonical TruD substrates and found that they are unable to complement an E. coli truD deletion or an S. cerevisiae pus7 deletion. Instead, the human PUS7 enzyme proved to be highly specific to the human tRNAGlu sequence, displaying activity targeted to U13. This activity appears to be independent of accessory factors. Taken together, this work strives to further our knowledge of Ψ synthases by examining TruD homologs present in higher organisms. TruD has the least sequence identity to the other four synthase families and does not possess any of the known RNA binding motifs. This work expands our knowledge base of the TruD family, the most divergent of the five Ψ synthase families.

La conversion des résidus U en pseudouridine (?) est la modification la plus fréquente dans les ARN. Cette réaction est catalysée par des ARN:?-synthases qui fonctionnent soit seules, soit au sein de particules snoRNP H/ACA composées de 4 protéines et d’un snoRNA. Nous avons démontré que l’ARN:?-synthase Pus7p de S. cerevisiae est capable d’agir sur les ARNt cytoplasmiques (position 13 dans 10 ARNt et position 35 dans le pré-ARNtTyr contenant un intron) (Behm-Ansmant et al., 2003). En parallèle, j’ai recherché les requis en séquence et en structure pour qu’un ARN soit substrat de Pus7p. J’ai également réalisé une étude préliminaire de l’importance des domaines additionnels de Pus7p par rapport à l’enzyme TruD d’E. coli. Comme les systèmes enzymatiques responsables de la formation de trois résidus ? du snRNA U2 de S. cerevisiae avaient été identifiés, nous avons pu débuter une étude de l’importance fonctionnelle de ces résidus pour la réaction d’épissage en utilisant l’approche globale par puces à ADN que l’équipe de J. Beggs (Edinburgh University) avait mise au point pour S. cerevisiae. Nous avons ainsi observé une baisse de l’efficacité d’épissage de certains ARN pré-messagers lorsque plusieurs gènes codant des U2 snRNA:?-synthases sont inactivés simultanément. L’ARN:?-synthase Pus7p est présente dans les 3 domaines du vivant et en particulier chez les archaea où l’activité de cette enzyme n’avait jamais été étudiée. Nous avons découvert l’existence de 2 systèmes enzymatiques différents pour la modification d’une même position d’un ARNt chez 2 espèces d’archaea thermophiles : une enzyme seule chez Pyrococcus abyssi contre un système de guide sRNP H/ACA chez Sulfolobus solfataricus dans lequel la protéine Pus7 est mutée au niveau d’acides aminés requis pour l’activité. Finalement, nous avons produit chacune de ces enzymes en grandes quantités et nous avons réalisé des tests de cristallisation. Des cristaux de Pus7 de P. abyssi ont été obtenus et une mesure complète de données de diffraction de rayons X a été réalisée au laboratoire à 2,5 Å de résolution. / thesis

Myotonic dystrophy is a genetic neurodegenerative disease caused by repeat expansion mutations. Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the 3’ UTR of the dystrophia myotonia protein kinase (DMPK) gene, while myotonic dystrophy type 2 (DM2) is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein nine (Znf9) gene. When expressed, these genes produce long CUG/CCUG repeat RNAs that bind and sequester a family of RNA-binding proteins known as muscleblind-like 1, 2 and 3 (MBNL1, MBNL2, MBNL3). Sequestration of these proteins plays a prominent role in pathogenicity in myotonic dystrophy. MBNL proteins regulate alternative splicing, and myotonic dystrophy symptoms are a result of mis-spliced transcripts that MBNL proteins regulate. MBNL proteins bind to a consensus sequence YGCY (Y = pyrimidine), which is found in CUG and CCUG repeats, and cellular RNA substrates that MBNL proteins bind and regulate. CUG and CCUG repeats can form A-form helices, however it is hypothesized that MBNL proteins bind to the helices when they are open and the YGCY binding site is single-stranded in nature. To evaluate this hypothesis, we used structure-stabilizing RNA modifications pseudouridine (Ψ) and 2’-O-methylation to determine if stabilization of CUG and CCUG repeat helices affected MBNL1 binding and toxicity. We also used Ψ to determine if the structure-stabilizing modification affected MBNL binding to single-stranded YGCY RNA. CUG repeats modified with Ψ or 2’-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Ψ also stabilized the structure of CCUG repeats and rigidified single-stranded YGCY RNA and inhibited MBNL1 binding to both of these RNAs. Binding data from CCUG repeats and single-stranded YGCY RNA suggest that both pyrimidines in the YGCY motif must be modified for significant inhibition. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Molecular dynamics simulations suggest that Ψ increases base-stacking interactions, and reducing the flexibility of single-stranded RNA leads to reduced MBNL1 binding. Ψ modification rescued mis-splicing in a cellular DM1 model and prevented CUG repeat toxicity in zebrafish embryos. This dissertation includes previously published and unpublished coauthored material.

No description available.

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Eugene G. Mueller, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

AN ABSTRACT OF THE DISSERTATION OF MANISHA DEOGHARIA, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry presented on May 16, 2018, at Southern Illinois University, Carbondale TITLE: PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs MAJOR PROFESSOR: DR. RAMESH GUPTA RNAs undergo several post-transcriptional modifications inside the cell. The most abundant modification found in RNA is pseudouridine. Pseudouridine is present in all major classes of RNA. The classical TΨC sequence of tRNA reflects T (ribothymidine or 5-methyluridine) at position 54 in most Bacteria and Eukarya, and Ψ and C at positions 55 and 56, respectively, in nearly all tRNAs. TrmA and TruB homologs produce T54 and Ψ55, respectively, in Bacteria and Eukarya. However, archaeal tRNAs commonly have Ψ54 (or m1Ψ54) instead of T54, and Pus10 produces both Ψ54 and Ψ55 in these tRNAs. The pus10 gene is present in nearly all Archaea and most eukaryotes, but not in Bacteria and yeast. This coincides with the presence of Ψ54 in archaeal tRNAs and certain tRNAs (for Gln, Trp, Pro Thr, etc.) of animals, and its absence in the tRNAs of Bacteria and yeast. tRNAs for Trp and Pro that function as primers for replication of retroviruses also contain Ψ54. We found that Pus10 is the Ψ54 synthase in eukaryotes. The Ψ54 activity is specific for certain tRNAs, and it requires a conserved Am1AAU sequence at positions 57-60 of the tRNA for its maximum activity. Recombinant Pus10 can also form Ψ54 in select tRNAs and presence of m1A at position 58 is necessary for its maximum activity. Humans have two paralogs of TruB, TruB1, and TruB2 which are predicted to be the Ψ55 synthases for cytoplasmic and mitochondrial tRNAs, respectively. We found that recombinant human Pus10 can also modify Ψ55 of tRNAs in vitro. This Ψ55 activity of human Pus10 is not selective for specific tRNAs. Another pseudouridine synthase, Cbf5, which functions in guide dependent manner, is necessary for Ψ production in 23S rRNA of H. volcanii. Cbf5 is the catalytic component of the box H/ACA ribonucleoprotein complex that brings about these modifications. It consists of a guide RNA and three core proteins Nop10, Gar1, and L7Ae along with Cbf5. We found that Nop10 is necessary for Ψ production in 23S rRNA.

No description available.

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the TψC stem. In the halophilic euryarchaeon Haloferax volcanii, tRNATrp and tRNAMet, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and ψ54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNATrp Cm34 formation being guided by its own intron, while that of tRNAMet being guided by a unique guide RNA called sR-tMet. We created genomic deletion of H. volcanii tRNATrp intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. ψ54, the near universal modification found in TψC stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, Methanocaldococcus jannaschii (MjPus10) and Pyrococcus furiosus (PfuPus10) have differential activities towards ψ54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA ψ54 formation. Finally, as an extension to this study, we have identified a novel tRNA ψ54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10.

No description available.