Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-119).

Conditional protein splicing (CPS) is an intein-mediated post-translational modification. Inteins are intervening protein elements that autocatalytically excise themselves from precursor proteins to ligate flanking protein sequences, called exteins, with a native peptide bond. Artificially split inteins can mediate the same process by splicing proteins in trans, when intermolecular reconstitution of split intein fragments occurs. An established CPS model utilizes an artificially split Saccharomyces cerevisiae intein, called VMA. In this model, VMA intein fragments are fused to the heterodimerization domains, FKBP and FRB, which selectively form a complex with the immunosuppressive drug, rapamycin. Treatment with rapamycin, therefore, heterodimerizes FKBP and FRB, and triggers trans-splicing activity by proximity association of intein fragments. Here, we engineered a CPS model to assemble inert fragments of the potent fungal ribotoxin, alpha (α)-sarcin, in vivo. Using this model, we demonstrate rapamycin-dependent protein splicing of α-sarcin fragments and a corresponding induction of cytotoxicity in HeLa cells. We further show that permissive extein context and incubation temperature are critical factors regulating the splicing of active target proteins. Ultimately, this technology could have potential applications in the fields of developmental biology and anti-tumour therapy.

Οι COUP-TFs είναι πυρηνικοί μεταγραφικοί παράγοντες, οι οποίοι υπάγονται στην υπεροικογένεια των πυρηνικών υποδοχέων. Τα μέλη της οικογένειας COUP-TF γενικά θεωρούνται καταστολείς της μεταγραφής και πολυάριθμοι μηχανισμοί έχουν προταθεί να υποστηρίζουν αυτή τους τη δράση. Με σκοπό να αποσαφηνιστεί ο μηχανισμός καταστολής των μελών της οικογένειας των COUP-TFs, χρησιμοποιήθηκε η μέθοδος yeast two-hybrid για να αναγνωριστούν πρωτεΐνες που εκφράζονται επίσης στον εγκέφαλο και ταυτόχρονα αλληλεπιδρούν άμεσα με μέλη της συγκεκριμένης οικογένειας. Η πρωτεΐνη CTIP1, ένα μέλος της καινούργιας οικογένειας των C2H2 πρωτεϊνών δακτύλου ψευδαργύρου, που απομονώθηκε σαν μία πρωτεΐνη που αλληλεπιδρά άμεσα με τον ARP1 και πιθανόν να εμπλέκεται στη μεταγραφική καταστολή που διαμεσολαβείται από τον ARP1, η οποία είναι ανεξάρτητη από την ευαίσθητη στην τριχοστατίνη Α αποακετυλίωση των ιστονών. Τόσο η CTIP1 όσο και η CTIP2 εκφράζονται σε μεγάλο βαθμό στον εγκέφαλο μια περιοχή για την οποία είναι γνωστό ότι εκφράζονται τα μέλη της οικογένειας των COUP-TFs, υπονοώντας ότι αυτή η καινούρια οικογένεια των πρωτεϊνών C2H2 δακτύλου ψευδαργύρου πιθανόν να εμπλέκεται στο σηματοδοτικό μονοπάτι των COUP-TFs. Στο πρώτο μέρος της παρούσας μεταπτυχιακής διατριβής, ο στόχος μας ήταν να διερευνήσουμε το πρότυπο έκφρασης της πρωτεΐνης COUP-TF Interactive Protein (CTIP) στα πρώιμα αναπτυξιακά στάδια του αχινού Paracentrotus lividus με τη μέθοδο του ανοσοφθoρισμού και να συγκρίνουμε το πρότυπο αυτό με το πρότυπο έκφρασης του COUP-TF και ταυτόχρονα να ελέγξουμε αν οι δύο αυτοί μεταγραφικοί παράγοντες αλληλεπιδρούν μέσω πειραμάτων ανοσοκατακρήμνισης. Ταυτόχρονα σημαντική ήταν και η προσπάθεια να μελετηθεί και η κατανομή των δύο ισομορφών του COUP-TF στα Μετάζωα. Το γονίδιο του COUP-TF αποτελείται, σε όλους τους μέχρι σήμερα μελετημένους οργανισμούς, από τρία εξώνια και δύο εσώνια που διακόπτουν την κωδική περιοχή του γονιδίου σε συντηρημένες θέσεις. Ωστόσο, έχει βρεθεί ότι ο αχινός Paracentrotus lividus διαφοροποιείται από αυτό το μοντέλο καθώς το γονίδιο αποτελείται από τέσσερα εξώνια (αδημοσίευτα αποτελέσματα του εργαστηρίου μας). Συγκεκριμένα, έχει βρεθεί ότι το επιπλέον εξώνιο βρίσκεται μεταξύ του πρώτου και του δεύτερου εξωνίου και έχει μέγεθος 63nt. Στο πρωτογενές μετάγραφο του COUP-TF συμβαίνει εναλλακτικό μάτισμα που έχει ως αποτέλεσμα την παραγωγή δύο m RNAs που κωδικοποιούν δύο πρωτεΐνες, οι οποίες διαφέρουν ως προς το μέγεθος λόγω της εισαγωγής 21 επιπρόσθετων αμινοξέων στην καρβόξυτελική περιοχή (CTE) της περιοχής πρόσδεσης στο DNA (DBD). Αν και πολλά δεν είναι γνωστά σχετικά με την ενδοκυτταρική τοποθέτηση κυρίως της μεγάλης ισομορφής, πειράματα EMSA ωστόσο εδειξαν οι δύο αυτές πρωτεΐνες διαφέρουν ως προς την ικανότητα πρόσδεσής τους στο DNA, με τη μεγάλη υπομονάδα του COUP να στερείται της ικανότητας να προσδένεται σε COUP στοιχείο απόκρισης. Σε μια προσπάθεια να μελετηθεί διεξοδικότερα το φαινόμενο του εναλλακτικού ματίσματος και συγκεκριμένα να διαπιστωθεί αν πρόκειται για ένα φαινόμενο συντηρημένο στα εχινοειδή, αποκλειστικό ή όχι αυτών, το συμπέρασμα που προέκυψε είναι ότι το εναλλακτικό μάτισμα είναι συντηρημένο και εμφανίζεται σταθερά στα είδη των αχινών που μελετήθηκαν (Paracentrotus lividus, Spaerechinus granularis, Strongylocentrotus purpuratus). Το εναλλακτικό μάτισμα λαμβάνει χώρα σε όλα τα στάδια από τα αγονιμοποίητα ωάρια εως τον πλουτέα και σε όλους τους ιστούς που o COUP-TF εκφράζεται (κοιλωματικά κύτταρα, μύες, έντερο). Για να μελετήσουμε την εξάπλωση του εναλλακτικού ματίσματος στα Μετάζωα, επιλέχθηκαν αντιπρόσωποι απ΄ όλες τις ομοταξίες του φύλου των Εχινόδερμων (Strongylocentrotus purpuratus, Amphiura filiformis, Patiria miniata, Marthasteria glacialis, Holothuria polii, Oxycomanthus japonicas), του φύλου των Ημιχορδωτών (Sakoglossus kowalevski), του φύλου των Χαιτόγναθων (Sagita minima), του υπόφυλου των Ουροχορδωτών (Ciona intestinalis) και του φύλου Xenoturbella (Xenoturbella bocki) για την απομόνωση ολικού RNA από καθέναν από αυτούς τους οργανισμούς. Αυτό το υλικό αποτέλεσε το υπόστρωμα για την πραγματοποίηση μιας σειράς πειραμάτων RT-PCR και Nested PCR με τη χρήση ειδικά σχεδιασμένων εκκινητών (degenerate primers). Η κλωνοποίηση των προιόντων σε κατάλληλο φορέα συνέβαλλε στην αλληλούχηση των κατάλληλων κλώνων. Παράλληλα, με το πρόγραμμα BLAST ψάξαμε για ομολογία της μικρής ισομορφής της πρωτεΐνης του COUP-TF του Paracentrotus lividus με άλλες πρωτεΐνες άλλων οργανισμών. Τα αποτελέσματα που προέκυψαν τόσο μέσα από τη νουκλεοτιδική αλληλούχηση όσο και μέσω της ηλεκτρονικής διερεύνησης συνηγορούσαν υπέρ της παρουσίας του εναλλακτικού ματίσματος ως ενός φαινομένου καθολικού που αφορά ευρύτερες (αν όχι όλες) ομάδες των Μεταζώων. / --

The mapping of biomolecular interactions reveals that the function of most biological components depends on a web of interrelations with other cellular components, stressing the need for a systems-level view of biological functions. In this work, I explore ways in which the integration of network and genomic information from different organizational levels can lead to a better understanding of cellular systems and components. First, studying yeast, I show that the evolutionary properties of target genes constitute the dominant determinant of transcription factor (TF) evolutionary rate and that this evolutionary modularity is limited to activating regulatory relationships. I also show that targets of fast-evolving TFs show greater evolutionary expression changes and are enriched for niche-specific functions and other TFs. This work highlights the importance of trans-regulatory network evolution in species-specific gene expression and network adaptation. Next, I show that genes either lost or gained across fungal evolution are enriched in TFs and have very different network and genomic properties than universally conserved genes, including, in sharp contrast to other networks, a greater number of transcriptional regulators. Placing genes in the context of their evolutionary life-cycle reveals principles of network integration of gained genes and evidence for the progressive network and functional marginalization of genes as an evolutionary process preceding gene loss. In the final chapter, I study how alternative splicing (AS)-driven expansion of human proteome diversity leads to system-level complexity through the AS-mediated rewiring of the protein-protein interaction network. By overlaying different network and genomic datasets onto the first large-scale isoform-resolution interactome, I found that differentiating between splice variants is essential to capturing the full extent of the network's functional modularity. I also discovered that AS-mediated rewiring preferentially affects tissue-specific genes and that topologically different patterns of rewiring have distinct functional consequences. Furthermore, I found that most rewiring can be traced to the AS of evolutionarily conserved sequence modules, which promote or block interactions and tend to overlap linear motifs and disrupt known domain-domain interactions. Together, this work demonstrates that a network-level perspective and genomic data integration are essential to understanding the evolution and functional diversity of proteomes.

The human genome is highly heterogeneous in its GC composition. How codon usage affects translation rates has been extensively studied and exploited to increase protein expression. Although effects on virtually all other steps in gene expression have been reported as well, so far no systematic approach has been taken to quantitatively measure the contribution of each to overall protein levels in human cells. Here, I utilise a library of several hundred synonymous variants of the Green fluorescent protein (GFP) to characterise the influence of codon usage on gene expression in human cells. In an initial small-scale screen, I show that protein levels are largely correlated with codon-usage and particularly GC-content. Additionally, I demonstrate that these changes can already be seen on the RNA level, confirming more broadly previously published data from our lab (Kudla et al., 2006). In order to assess the consequences of randomised codon usage on a larger scale, I established and validated a high-throughput approach for the phenotypic profiling of reporter genes. Using a pool of cells stably expressing >200 GFP variants, I measured multiple parameters simultaneously, such as protein levels, translational state, RNA levels, stability and export. Data from these experiments confirm a strong relationship between GC-content, protein levels, as well as RNA export, reproducibly in two cell lines. Low expression of especially GC-poor variants could not be rescued by splicing, but increased nuclear-to-cytoplasmic RNA ratio, suggesting further mechanisms important for efficient gene expression. These effects are even more pronounced when the distribution of GC is spread evenly along the coding sequence. Interestingly, our data also suggests that high GC within the first 200nt is more predictive of efficient gene expression, contrasting studies performed on bacteria, in which strong secondary folding near the ribosomal binding site was shown to be non-permissive for translation (Kudla et al., 2009). By relating experimentally derived parameters to sequence features known to inhibit expression, I demonstrate that cryptic splicing is a major factor leading to decreased levels of particularly GC-poor GFP variants. An attempt to quantitatively assess the relative contribution of several sequence features (e.g. tAI, GC3, CpG) using multiple regression analysis lead to inconclusive results, leaving the requirement for the exploration of alternative approaches in order to dissect the role of individual parameters, as well as to identify novel determinants of gene expression.

I investigated two aspects of in vivo splicing that are poorly understood: spliceosome disassembly and recycling, and proofreading. To this end, I used the auxin-inducible degron (AID) to individually deplete several splicing factors in budding yeast and then I measured the effect on co-transcriptional spliceosome assembly through chromatin immunoprecipitation. In addition, using RNA next-generation sequencing, I measured the frequency of splicing errors following depletion or mutation of the fidelity factor, Prp22. I show that formation of the pre-spliceosome (the first stage of spliceosome assembly) is rapidly inhibited by global defects in late stages of spliceosome assembly. I demonstrate that this is due to the accumulation of arrested spliceosomes that sequester the splicing machinery and, as a result, causes a recycling defect. This suggests that spliceosomes that lack essential splicing factors are not always properly disassembled and recycled in vivo, and warns about potential systematic secondary effects when perturbing single components of the spliceosome. Secondly, I describe the development of a new version of the AID system for budding yeast, called the B-estradiol AID. To the best of my knowledge, an AID system for budding yeast that is fast-acting, tightly-controlled and gratuitous, was lacking until now. Lastly, I show that absence of Prp22 protein, which was previously proposed to play a role in splicing fidelity, correlates with more mistakes in 3’ss selection of many endogenous intron-containing transcripts in vivo. This provides indirect evidence to suggest that Prp22-dependent splicing proofreading is physiologically important. The data from this analysis will be useful in ongoing studies to try to identify common features that could improve our understanding of the mechanism of Prp22’s function in splicing proofreading.

Le Gallid Herpesvirus 2 (GaHV-2) est un herpesvirus responsable de lymphomes T chez le poulet. Son génome code pour 25 miARN matures regroupés en 2 clusters localisés au sein des régions répétées TRL/IRL etIRS/TRS. Mon travail a tout d’abord consisté en l’étude de la fonctionnalité des deux brins de mdv1-pré-miRM4sur deux cibles virales UL-28 et UL32. Le brin mdv1-miR-M4-5P ayant été identifié comme orthologue dumiARN cellulaire miR-155, leurs impacts ont été comparés sur six cibles cellulaires et la cible virale UL-28.Les différents miARN de GaHV-2 présentant des taux d’expression variables, nous avons identifié, dans larégion TRL/IRL, des ARNm responsables de leur transcription à partir d’exon ou d’intron. De plus, nous avonsidentifié un promoteur plutôt spécifique de la latence et responsable de la transcription de l’ensemble desmiARN de l’IRL/TRL, de l’oncogène meq et du transcrit Meq/vIL-8. / Gallid Herpesvirus 2 (GaHV-2) is a herpesvirus inducing T-lymphoma in chickens. Its genome encodes 25mature miRNAs splitted in two clusters localized in IRL/TRL and IRS/TRS repeated regions. In this work, wefirst analyzed the functionality of the two strands of mdv1-pre-miR-M4 on two viral targets named UL-28 andUL-32. As one strand of this miRNA was identified as an ortholog of cellular miRNA-155, their effects wereassessed on six cellular targets and on the UL-28 viral target. As a differential expression of miRNAs wasobserved during GaHV-2 infection, we found numerous IRL/TRL transcripts responsible for the intronic orexonic transcription of viral miRNAs. Moreover, we identified a latency promoter controlling at the same timetranscription of all IRL/TRL miRNAs, the oncogenic meq gene and the Meq/v-IL8 transcript.

xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the protein MBNL1 (M[barbelow]uscleb[barbelow]lin[barbelow]d-l[barbelow]ike-1) is sequestered to the expanded CUG or CCUG RNAs. MBNL1 regulates the alternative splicing of a variety of pre-mRNAs and its mis-localization results in mis-splicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. I initially demonstrated that MBNL1 can bind short structured CUG and CCUG repeats with high affinity and specificity in vitro . Next, I was able to determine and articulate the first structure of a binding site of MBNL1 in an endogenous pre-mRNA that it regulates. I found that MBNL1 binds a stem-loop in the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. I determined that MBNL1 regulated exon 5 by directly competing with the essential splicing factor U2AF65 for binding upstream of exon 5. When U2AF65 is prevented from binding, factors in the spliceosome can no longer be recruited and the following exon is skipped. Furthermore, I found that MBNL1 and U2AF65 compete by binding mutually exclusive RNA structures. I also characterized a potential therapeutic approach for DM. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. Using a targeted screen of small molecules known to bind structured nucleic acids, I identified the small molecule pentamidine as a compound that disrupted MBNL1 binding to CUG repeats in vitro . I showed in cell culture that pentamidine was able to reverse the mis-splicing of two pre-mRNAs affected in DM. Pentamidine also significantly reduced the formation of RNA foci in tissue culture cells, which are characteristic of DM. MBNL1 was released from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of two pre-mRNAs in mice expressing expanded CUG repeats. This dissertation includes three previously published co-authored publications. / Committee in charge: Kenneth Prehoda, Chairperson, Chemistry; J. Andrew Berglund, Advisor, Chemistry; Victoria DeRose, Member, Chemistry; Peter von Hippel, Member, Chemistry; Alice Barkan, Outside Member, Biology

Made available in DSpace on 2015-07-22T20:49:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-28 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / ABSTRACT:The measurement of serum thyroglobulin(sTg)has been considered the most sensitive tumor marker in the follow-up of patients with differentiated thyroid carcinoma (DTC) after total thyroidectomy and radioiodine therapy. However, sTg assays present some technical problems, specially, the interference by endogenous anti-thyroglobulin antibodies(TgAb), present in approximately 20 por cento of DTC patients' sera. Therefore, in order to enhance sensitivity and to hinder the interference by TgAb, several investigators have tried to quantify the mRNA Tg by Real-Time RT-PCR. However, the results have been variable and some of them did not report a correlation between mRNA Tg measurement and presence of metastases.The aim of this study was to evaluate TgmRNA expression, performed by a new sensitive and specific Real-Time PCR, in peripheral blood of 104 patients who previously undergone total thyroidectomy and radioiodine treatment. DTC patients were studied during L-T4 therapy.Eighty-two patients out of 104(78.8 por cento)were considered “free of disease”, and 22(21.2 por cento) presented metastases.The quantification of mRNA was significantly different between patients “free of disease”and those with metastases:TgmRNA(90.1 vs 951.3 ug/uL)(P<0,0001).In conclusion, the differences proposed in the TgmRNA quantification made it a reliable method, and allowed us to differentiate patients free of disease from patients with metastases and thus could be an appropriate molecular marker in the follow-up of patients with thyroid carcinoma, especially in patients with positive TgAb.. / FAPESP: 04/09934-7 / FAPESP: 05/55842-0 / TEDE / BV UNIFESP: Teses e dissertações