Dissertations / Theses on the topic 'MRNA profiling'

<p> The RNA-binding proteins U2AF and PTB play important roles in gene expression in many eukaryotic species. Although U2AF and PTB have been well-studied, their functional requirements have not been investigated on a genome-wide scale. In this thesis, I analyze RNA expression data to determine the requirement of the general splicing factor U2AF in <i>S. pombe</i> and to identify genes misregulated in Drosophila PTB mutants. I find that many introns are insensitive to U2AF inactivation in a <i>Schizosaccharomyces pombe</i> U2AF59 mutant, <i>prp2.1.</i> Bioinformatics analysis indicates that U2AF-insensitive introns have stronger 5' splice sites and higher A/U composition. The importance of intronic nucleotide composition was further investigated using wild type RNA expression data sets. I show that nucleotide composition is a relatively important factor for regulated intron retention in a variety of species. I also analyzed the RNA-binding protein PTB using RNA Seq data to reveal genes misregulated in PTB mutants in <i>D. melanogaster.</i> I identify misregulation of alternative splicing in PTB mutants and putative PTB binding sites. In the PTB embryonic lethal mutant, which shows dorsoventral patterning defects, I show that dorsal fate genes are significantly up-regulated. I present a model to link PTB to dorsal closure defects. This thesis provides the first genome-wide analysis of U2AF in <i>S. pombe</i> and PTB in <i>Drosophila melanogaster. </i></p>

La régulation dynamique de la synthèse des protéines en fonction des besoins de la cellule facilite son adaptation face aux fluctuations de l’environnement. Malgré l’importance de la régulation de la traduction au cours du processus d’expression des gènes, l’impact de ce mécanisme sur des processus biologiques fondamentaux, comme la mise en place d’une réponse immunitaire, reste mal compris. Grâce au développement de nouvelles technologies basées sur l’utilisation du séquençage à haut débit, comme le ribosome profiling, il est désormais possible d’étudier en détails la façon dont la synthèse des protéines est contrôlée. Le monosome vs polysome footprinting est une nouvelle méthode qui permet d’étudier la traduction des ARN messagers (ARNm) selon leur association avec un seul ribosome (monosome) ou avec plusieurs ribosomes (polysomes). Au cours de ma thèse, j’ai identifié les paramètres essentiels pour la mise en place d’une expérience de monosome vs polysome footprinting donnant des résultats fiables en utilisant des macrophages primaires dérivés de la moëlle osseuse de souris. Je me suis intéressée à ce type de cellules immunitaires particulier car elles présentent une grande capacité à détecter des modifications dans leur environnement et à modifier leur taux d’expression de protéines en fonction des signaux reçus. Leur grande plasticité est notamment essentielle pour assurer leurs diverses fonctions de protection de l’organisme, comme le déclenchement et la résolution de la réponse inflammatoire. La méthode de monosome vs polysome footprinting ayant été initialement développée chez la levure, son utilisation avec un modèle d’étude différent a nécessité de nombreuses modifications du protocole. Suite à cette phase de développement technologique, j’ai pu confirmer que les monosomes, une population de ribosomes historiquement considérés comme inactifs, sont activement impliqués dans le processus de traduction dans les macrophages primaires de souris. Les données obtenues ont également permis d’identifier des caractéristiques communes entre les ARNm enrichis dans les monosomes chez la levure et dans les macrophages murins. La régulation de la synthèse des protéines via l’association à des monosomes ou à des polysomes pourrait donc être un mécanisme conservé chez les organismes eucaryotes. Enfin, le travail d’optimisation réalisé dans les macrophages primaires murins ouvre la possibilité d’étudier l’effet de la régulation de la traduction sur la mise en place et la résolution de la réponse inflammatoire de façon très détaillée<br>The dynamic regulation of the protein synthesis process participates in the cell adaptation to a constantly evolving environment. Despite its critical role in gene expression regulation, the understanding of translational control in fundamental biological processes, such as immune responses, is still incomplete. The implementation of new approaches based on deep sequencing can be used to fill the gap in the knowledge of protein synthesis regulation. Notably, monosome vs polysome footprinting is an innovative approach derived from ribosome profiling that allow the characterization of 80S footprints derived either from monosomes or polysomes associated ribosomes. In this work, I identified the key parameters required to obtain a robust picture of ribosomal densities across cellular mRNAs using monosome vs polysome footprinting in murine primary bone-marrow derived macrophages (pBMDM). These immune cells are particularly interesting to study protein synthesis regulation in evolving conditions as they display a high sensitivity towards their environment and have the ability to trigger different gene expression programs depending on external cues. Their high phenotypic plasticity is in fact essential to ensure their protective functions in the organism such as the triggering and the resolution of the inflammatory response. As monosome vs polysome footprinting was initially developed in yeast, the adaptation of this method to study murine immune cells required extensive optimizations. The resulting protocol developed in this work was used to confirm that, contrary to a long lasting belief in the scientific community, murine pBMDM monosomes are actively involved in the translation process. Interestingly, we were able to recapitulate similar observations to what was previously observed in yeast regarding the features of mRNAs preferentially bound to monosomes or polysomes in murine pBMDM. This could suggest that the differential trafficking of ribosomes depending on specific features of the cellular mRNAs is a conserved mechanism of translational control. Importantly, the distribution of ribosomes across the different mRNAs is not random and the proper ribosome allocation pattern could be critical to adapt protein synthesis levels to the cellular needs. Here we developed a robust strategy to study this overlooked transcript-specific mechanism of translational control. Moreover, our optimized protocol can now be used to study the impact of translation through monosomes or polysomes at different stages of the inflammatory response in murine macrophages

The exonuclease Xrn1 degrades messenger RNAs (mRNAs) and controls transcription. By using a model system that replicates brome mosaic virus (BMV) in yeast, here we show that Xrn1 promotes translation initiation of BMV RNA. This function is linked to the highly structured 5’UTR and the coding sequence. The exonuclease activity of Xrn1 is required for its role in viral RNA translation. In contrast, the role of Xrn1 in translation is independent from its function in transcription. Importantly, ribosome profiling analyses reveal that Xrn1 acts as a translational activator of a specific group of cellular mRNAs enriched for glycosylation functions. As for BMV RNA, these cellular mRNAs depend on the exonuclease activity of Xrn1 for translational activation and contain long and structured 5’UTR. Our results reveal a new cross-talk between mRNA degradation and translation, and uncover an unexpected function of the exonuclease Xrn1 in viral and cellular translational control. Xrn1 és una exonucleasa que degrada l’ARN missatger (ARNm) i en regula la transcripció. Emprant un sistema model basat en el virus del mosaic del brom (BMV) i el llevat S. cerevisiae, en aquest treball es demostra que Xrn1 promou l’inici de la traducció de l’ARN viral. Aquesta funció en traducció està lligada a la regió no traduïda a l’extrem 5’ i a la seqüència codificant. L’activitat exonucleasa pròpia de Xrn1 és necessària per tal que actuï en traducció. En canvi, la funció de Xrn1 en transcripció és independent de la funció en traducció. Mitjançant estudis amb perfil de ribosomes, es va constatar que Xrn1 també actua com a activador de la traducció d’un subgrup d’ARNm cel·lulars. Aquests, codifiquen per funcions relacionades amb la glucosilació. Tal i com s’observa per l’ARN de BMV, aquests ARNm cel·lulars depenen de l’activitat exonucleasa de Xrn1 per a la seva traducció i tenen una elevada estructura secundària a la regió no traduïda de l’extrem 5’. En conjunt, els nostres resultats descriuen un nou exemple de la comunicació existent entre la degradació i la traducció de l’ARNm i revelen que Xrn1 té una funció inesperada en el control de la traducció de l’ARNm viral i cel·lular.<br>Xrn1 és una exonucleasa molt conservada entre espècies i que té un paper cabdal en la degradació de l’ARN missatger (ARNm) i la regulació de la transcripció. Emprant un sistema model basat en el virus del mosaic del brom (BMV) i el llevat Saccharomyces cerevisiae,  en  aquest  treball  es  demostra  que  Xrn1  promou  la  traducció  de  l’ARN     viral. Aquesta funció en traducció està lligada a la regió no traduïda a l’extrem 5’ i a la seqüència codificant. A més, mitjançant l’anàlisi amb perfils de polisomes s’observa que  Xrn1  assisteix  la  iniciació  de  la  traducció  de  l’ARN  viral  i  que  interacciona           directament amb la maquinària de traducció. Tenint en compte que Xrn1 par􀀁cipa en la degradació i la transcripció d’ARNm, es van crear diferents mutants per tal d’analitzar la relació entre aquestes funcions i el rol de Xrn1 en traducció. En primer lloc, es va observar que l’ac􀀁vitat exonucleasa pròpia de Xrn1  és  necessària  per  tal  que  Xrn1  actuï  en  traducció.  Expressant  la  exonucleasa    nuclear (Rat1) al citoplasma de cèl·lules xrn1∆, s’aconsegueix compensar els defectes en degradació de l’ARN i en la taxa de creixement. En canvi, no es rescaten els defectes en la traducció de l’ARN viral. En segon lloc, u􀀁litzant un mutant de Xrn1 que no pot ser importat al nucli (Xrn1∆NLS) es va concloure que la funció de Xrn1 en transcripció és independent de la funció en traducció. Tot seguit, mitjançant estudis amb perfil de ribosomes, es va constatar que Xrn1 també actua com a ac􀀁vador de la traducció  d’un  subgrup  d’ARNm  cel·lulars.  Aquests,  codifiquen  per  funcions              relacionades amb la glucosilació i estan enriquits en proteïnes del re􀀁cle endoplasmà􀀁c. Tal i com s’observa per l’ARN de BMV, aquests ARNm cel·lulars depenen  de  l’ac􀀁vitat  exonucleasa  de  Xrn1  per  a  la  seva  traducció  i  tenen  una         elevada estructura secundària a la regió no traduïda de l’extrem 5’. En  conjunt,  els  nostres  resultats  descriuen  un  nou  exemple  de  la  comunicació         existent entre la degradació i la traducció de l’ARNm i revelen que Xrn1 té una funció inesperada en el control de la traducció de l’ARNm viral i cel·lular.

Protein synthesis is a fundamental and ultimate process in living cells. Cells possess sophisticated machineries and continuously carry out complex processes. Monitoring protein synthesis in living cells not only inform us about the mechanism of translation but also deepen our insights about all aspects of life. Understanding the structure and mechanism of the ribosome and its associated factors helped us enlarge our knowledge on protein synthesis. Recently, with the dramatic advances of high-throughput sequencing and bioinformatics, a new technique called ribosome profiling emerged. By retrieving mRNA fragments protected by translating ribosomes, ribosome profiling reveals global ribosome occupancy along mRNAs in living cells, which can inform us with the identity and quantity of proteins being made. Easily adapted to other organisms, ribosome profiling technique is expanding its application in revealing various cellular activities as well as the knowledge on protein synthesis. Here, we report the mechanism of translating mRNA cleavage by endoribonuclease RelE in vivo. RelE is an endoribonuclease that is induced during nutrient deficiency stress and specifically cleaves translating mRNAs upon binding to the ribosomal A site. Overexpression of RelE in living cells causes growth arrest by inhibiting global translation. We monitored RelE activity in vivo upon overexpression using ribosome profiling. The data show that RelE actively cuts translating mRNAs whenever the ribosomal A site is accessible, resulting in truncated mRNAs. RelE causes the ribosome complexes to accumulate near the 5' end of genes as the process of ribosome rescue, translation, and cleavage by RelE repeats. RelE cleavage specific sub-codon level ribosome profiling data also represent reading frame in Escherichia coli and sequence specificity of RelE cleavage in vivo. We report another ribosome profiling study on a methyltransferase TrmD in E. coli. TrmD is known to methylate G37 (the residue at 3' side of anticodon) of some tRNAs and be responsible for codon-anticodon interaction. We constructed a TrmD depletion E. coli strain, whose deletion results in lethality of cells. Resulting depletion of m1G37 in the strain leads to growth arrest. Lack of m1G37 of some tRNAs whose codons start with C showed frequent frameshift when translating the gene message in vitro. By using ribosome profiling, we successfully observed significant difference on translation process when codons interact with anticodons of tRNAs lacking m1G37. The data reveal slow translation rate or pauses on the tRNAs when missing the appropriate methylation, which corresponds to the previous biochemical data in vitro.

Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.<br>Ph.D.<br>Department of Chemistry<br>Burnett College of Biomedical Sciences<br>Biomolecular Sciences: Ph.D.

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.

The gastrointestinal tract (GIT) regulates nutrient uptake, secretes hormones and has a crucial gut flora and enteric nervous system. Of relevance for these functions are the G protein-coupled receptors (GPCRs) and the solute carriers (SLCs). The Adhesion GPCR subfamily is known to mediate neural development and immune system functioning, whereas SLCs transport e.g. amino acids, fatty acids (FAs) and drugs over membranes. We aimed to comprehensively characterize Adhesion GPCR and SLC gene expression along the rat GIT. Using qPCR we measured expression of 78 SLCs as well as all 30 Adhesion GPCRs in a twelve-segment GIT model. 21 of the Adhesion GPCRs had a widespread (≥5 segments) or ubiquitous (≥11 segments) expression. Restricted expression patterns were characteristic for most group VII members. Of the SLCs, we found the majority (56 %) of these transcripts to be expressed in all GIT segments. SLCs were predominantly found in the absorption-responsible gut regions. Both Adhesion GPCRs and SLCs were widely expressed in the rat GIT, suggesting important roles. The distribution of Adhesion GPCRs defines them as a potential pharmacological target. FAs constitute an important energy source and have been implicated in the worldwide obesity increase. FAs and their ratios – indices for activities of e.g. the desaturase enzymes SCD-1 (SCD-16, 16:1n-7/16:0), D6D (18:3n-6/18:2n-6) and D5D (20:4n-6/20:3n-6) – have been associated with e.g. overall mortality and BMI. We examined whether differences in FAs and their indices in five lipid fractions contributed to obesity susceptibility in rats fed a high fat diet (HFD), and the associations of desaturase indices between lipid fractions in animals on different diets. We found that on a HFD, obesity-prone (OP) rats had a higher SCD-16 index and a lower linoleic acid (LA) proportions in subcutaneous adipose tissue (SAT) than obesity-resistant rats. Desaturase indices were significantly correlated between many of the lipid fractions. The higher SCD-16 may indicate higher SCD-1 activity in SAT in OP rats, and combined with lower LA proportions may provide novel insights into HFD-induced obesity. The associations between desaturase indices show that plasma measurements can serve as proxies for some lipid fractions, but the correlations seem to be affected by diet and weight gain.

La FSH est une des hormones clés qui régule la reproduction chez les mammifères. Chez le mâle, elle cible les cellules de Sertoli, qui expriment le RFSH. La cellule de Sertoli a un rôle trophique important pour le bon développement de la spermatogenèse. Dans cette thèse, nous avons établi le premier traductome, c’est-à-dire l’ensemble des ARNm en cours de traduction, dépendant du RFSH. La traduction de certains ARNm significativement modulés par la FSH exercerait un rétrocontrôle sur la signalisation FSH-dépendante. L’analyse du protéome nous a permis de valider ce traductome au niveau systémique. Nous avons également démontré l’implication des β-arrestines dans la traduction d’ARNm dépendante de la FSH. Les β-arrestines forment un assemblage moléculaire avec le module de traduction p70S6K/rpS6. Cet assemblage est impliqué dans la traduction des ARNm 5’TOP, qui encodent la machinerie traductionnelle. C’est l’activation FSHdépendante des protéines G qui promeut l’activation de p70S6K au sein du module β-arrestines/ p70S6K/ rpS6. Ce travail constitue une nouvelle avancée sur les mécanismes grâce auxquels la FSH exerce sa fonction biologique de dans ses cellules-cibles naturelles de la gonade mâle<br>FSH is one of the key hormones that regulate the reproductive function in mammals. In the male, FSH targets Sertoli cells, which express the FSHR. Sertoli cells play an important trophic role in the development of spermatogenesis. Here, we have provided the first FSHR-induced translatome, that encompasses all the mRNA being actively translated. The translation of some mRNAs significantly modulated by FSH may exert a feedback control on FSH-dependent signaling. The analysis of the proteome has validated the FSHR translatome at the systems level. We also demonstrated the involvement of β-arrestins in the FSH-stimulated translation of mRNA. β-arrestins form a molecular assembly with the p70S6K / rpS6 translation module. This molecular assembly is involved in the translation of 5'TOP mRNA, which encode proteins of the translational machinery. FSH-activated G proteins leads to p70S6K activation within the β-arrestins/ p70S6K/ rpS6 module. This work provides new advance on the mechanisms whereby FSH exerts its biological function in its natural target cells of the male gonad

Magister Pharmaceuticae - MPharm<br>The growing resistance of Methicillin-Resistant Staphylococcus aureus (MRSA) to currently prescribed drugs has resulted in the failure of prevention and treatment of different infections caused by the superbug. Therefore, to keep pace with the resistance, there is a pressing need for novel antimicrobial agents, especially from non-conventional sources. Several natural products (NPs) have displayed varying in vitro activities against the pathogen but few of these natural compounds have been studied for their prospects to be potential antimicrobial drug candidates. This may be due to the high cost, tedious, and time-consuming process of conducting the important preclinical tests on these compounds. Hence, there is a need for cost-effective strategies for mining the available data on these natural compounds. This would help to get the knowledge that may guide rational prioritization of “likely to succeed” natural compounds to be developed into potential antimicrobial drug candidates.

Nicotine dependence is responsible for perpetuating the adverse health effects due to tobacco use, the leading cause of preventable death worldwide. Nicotine is an agonist for nicotinic acetylcholine receptors, which are enriched in the mesocorticolimbic and habenulo-interpeduncular circuitries, underlying nicotine reward and withdrawal, respectively. Drugs of abuse, including nicotine, induce stable neuroadaptations, requiring protein synthesis through regulation of transcription factors, epigenetic mechanisms, and non-coding RNAs. It also been shown that miRNAs in brain are regulated by nicotine and that miRNA dysregulation contributes to brain dysfunction, including drug addiction. While much is known about the neurocircuitry responsible for the behaviors associated with nicotine reward or withdrawal, the underlying molecular mechanisms of how these changes in behavior are induced are less clear. Using miRNA-/mRNA-Seq, we demonstrate that there are widespread changes in both miRNA and mRNA expression in brain regions comprising the mesocorticolimbic circuit after chronic nicotine treatment, and the habenulo-interpeduncular circuit during acute nicotine withdrawal. Conserved, differentially expressed miRNAs were predicted to target inversely regulated mRNAs. We determined that expression of miR-106b-5p is up-regulated and Profilin 2 (Pfn2), an actin-binding protein enriched in the brain, is down-regulated in the interpeduncular nucleus (IPN) during acute nicotine withdrawal. Further we show that miR-106b-5p represses Pfn2 expression. We demonstrate that knockdown of Pfn2 in the IPN is sufficient to induce anxiety, a symptom of withdrawal. This novel role of Pfn2 in nicotine withdrawal-associated anxiety is a prime example of this dataset’s utility, allowing for the identification of a multitude of miRNAs/mRNA which may participate in the molecular mechanisms underlying the neuroadaptations of nicotine dependence.

Neurotrophins and their receptors are key molecules in the development of the<br/>nervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor<br/>NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive<br/>form (150 kDa) and a truncated non-catalytic form (50 kDa). The two<br/>variants show different 3'UTR regions, indicating that they might be differentially<br/>regulated at the post-transcriptional level. In this work we explore how<br/>microRNAs take part in the regulation of full-length and truncated NTRK3,<br/>demonstrating that the two isoforms are targeted by different sets of microRNAs.<br/>We analyze the physiological consequences of the overexpression of some of the<br/>regulating microRNAs in human neuroblastoma cells. Finally, we provide<br/>preliminary evidence for a possible involvement of miR-124 - a microRNA with no<br/>putative target site in either NTRK3 isoform - in the control of the alternative<br/>spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.<br>Las neurotrofinas y sus receptores constituyen una familia de factores cruciales<br/>para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función<br/>principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual<br/>se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo<br/>tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas<br/>no comparten la misma región 3'UTR, lo que sugiere la existencia de una<br/>regulación postranscripcional diferente. En el presente trabajo se ha explorado<br/>como los microRNAs intervienen en la regulación de NTRK3, demostrando que las<br/>dos isoformas son reguladas por diferentes miRNAs. Se han analizado las<br/>consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando<br/>células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del<br/>microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la<br/>regulación de represor de splicing PTBP1.

Die vorliegende Arbeit beschäftigt sich mit der Frage, inwieweit quantitative Genexpressionsstudien an postmortalen humanen Proben mit erhöhtem Postmortalintervall und somit möglicherweise verminderter RNA-Integrität realisierbar sind und in Zukunft als zusätzliches diagnostisches Werkzeug zur Determinierung der Todesursache im forensischen Kontext herangezogen werden können. Dafür wurden in mehreren Teilstudien Faktoren untersucht, die die Verlässlichkeit quantitativer Genexpressionsdaten beeinflussen können. Es konnte zunächst für postmortales Skelettmuskelgewebe festgestellt werden, dass Verstorbene mit erhöhtem BMI statistisch signifikant niedrigere RIN-Werte aufweisen als normalgewichtige Personen. Zudem wurde eine Korrelation zwischen dem Gewebetyp und der Integrität der daraus extrahierten RNA gefunden. Unter Anwendung der in dieser Arbeit gewählten Extraktionsmethode scheint postmortales Skelettmuskelgewebe für Genexpressionsstudien an Autopsiematerial besonders geeignet. Dagegen wurde im vorliegenden Probengut kein Zusammenhang zwischen verminderter RNA-Integrität und Parametern wie Geschlecht, PMI, Sterbealter, Dauer der Agonie und Todesursache gefunden. In einer weiteren Teilstudie wurde anhand von postmortalem Herzmuskel-, Skelettmuskel- und Gehirngewebe aus einer Auswahl von zehn funktionell verschiedenen endogenen Kontrollgenen HMBS, UBC, SDHA und TBP als die Gene mit der größten postmortalen Transkriptstabilität identifiztiert. Zudem konnte gezeigt werden, dass die Verwendung von vier stabilen endogenen Kontrollen am untersuchten Probenmaterial eine verlässliche Datennormalisierung erlaubt. Die validierten Kontrollgene können auch zukünftig für quantitative Genexpressionsstudien eingesetzt werden, solange die untersuchte Probenzusammensetzung der hier vorgestellten ähnelt. Für die Todesursache sowie für den Body Mass Index des Probenspenders wurde in der vorliegenden Arbeit ein statistisch signifikanter Einfluss auf das Expressionslevel instabiler Gene festgestellt. Diese Parameter sind daher geeignet, die gefundenen Instabilitäten möglicher Kontrollgene zu erklären. Die Ergebnisse der vorliegenden Arbeit lassen des Weiteren darauf schließen, dass das Erstellen von Degradierungslinien aus kommerziell erhältlicher RNA für jedes verwendete qPCR-Assay ein wichtiges Instrument der Qualitätsprüfung ist. Mit der Degradierungslinie kann die Detektionsgrenze des verwendeten qPCR-Assays validiert werden kann. Nur so ist die Festlegung des Bereichs möglich, in dem ein verändertes Expressionslevel tatsächlich mit dem Einfluss eines spezifischen Parameters in Verbindung gebracht werden kann und klar von einer nur scheinbaren Genexpressionsänderung unterscheidbar ist, die durch eine Degradierung der Probe vorgetäuscht wird. Zudem scheint es praktikabel, in zukünftigen Studien nur Proben mit ähnlichen Integritäten miteinander zu vergleichen. Pathologische Prozesse im menschlichen Körper, auch solche, die die Funktion von Organen sowie die Morphologie betreffen, sind hoch komplex und können interindividuell variieren, weshalb die Genexpression im forensischen Kontext ergänzende Hinweise auf die Diagnose liefern könnte. Als erstes anwendungsbezogenes Beispiel wurde in der vorliegenden Arbeit der Einfluss von Hypoxie auf das Transkriptlevel von HIF-1α, VEGF und SLC2A1 untersucht. Bei Normalisierung der Daten gegen vier stabile Kontrollgene ergaben sich anhand der untersuchten Gewebeproben Hinweise auf eine todesursachenbezogene Hochregulierung der drei Zielgene. Die Studie unterstrich die besondere Bedeutung der gewählten Normalisierungsstrategie. Wurden die Daten nur gegen GAPDH als einzelnes, nicht validiertes Kontrollgen normalisiert, deuteten die Ergebnisse eine überraschende Herunterregulierung der Zielgene an. Als mögliche Ursache für diese scheinbare Diskrepanz kommt die im weiteren Verlauf dieser Studie nachgewiesene Instabilität infolge einer Co-Regulation von GAPDH unter hypoxischen Bedingungen in Betracht. Mit dem Fernziel postmortale Genexpressionsstudien als zusätzliches Instrument in der forensischen Todesursachenbestimmung einzusetzen, schafft die vorliegende Arbeit durch die Einführung von validierten Kontrollgenen sowie durch die Analyse weiterer Einfluss nehmender Faktoren eine Basis für die verlässliche Durchführung künftiger Genexpressionsstudien an humanem Autopsiegewebe<br>Using quantitative gene expression studies could form a valuable innovative tool for the determination of the cause of death in forensic pathologies. Thus, the present work focuses on the questions around the possibilities and limitations of gene expression analysis in postmortem human tissue from donors with prolonged postmortem interval (PMI) and probably decreased RNA integrity. Therefore several parameters influencing the reliability of quantitative gene expression data were examined. First, it was found that in postmortem skeletal muscle deceased with an increased body mass index (BMI) reveal decreased RIN values compared to normal weight donors. Moreover, a correlation was found between the type of tissue and the integrity of RNA that was isolated from it. When extracting RNA as presented in this work, postmortem skeletal muscle tissue showed the overall highest RIN values and seems to be best suited for gene expression studies using autopsy material. Using the presented sample set no correlation was found between RNA integrity and parameters like gender, PMI, age at death, duration of agony and cause of death. Second, using postmortem cardiac muscle, skeletal muscle and brain tissue the stability of ten endogenous control genes with different functionality was assessed and HMBS, UBC, SDHA and TBP were identified as genes of high transcript stability. Furthermore, it was shown that four stable endogenous control genes seem to be adequate for a reliable data normalisation. Thus, the four validated control genes mentioned may be used in future quantitative gene expression studies if sample composition is similar to the one presented in this work. Donors cause of death and body mass index were found to significantly influence the expression level of instable genes. Thus, these parameters may be adducted to explain the instability found in some of the assessed potential reference genes. Third, identifying the limit of detection due to degradation processes was found to be an important quality control test for every qPCR assay used. The results of this work confirm the importance of analyzing degradation lines of commercially available RNAs. This information allows the correct interpretation of gene expression levels and an identification of apparent changes in transcript abundances which are the result of degradation rather than biological processes. Finally, it was found that even though it is not always possible to collect only samples with high RIN values, it seems to be workable to use RNA samples of similar integrities in future gene expression studies. Fourth, pathological processes in the human body affecting the function of organs as well as their morphology show high complexity and may vary between individuals. Therefore, analysis of the gene expression status at the time point of death might give valuable hints about donors´ forensically relevant diagnose. Examining the impact of hypoxia on expression levels of HIF-1α, VEGF and SLC2A1 was conducted as a first example. Normalising data against four stable reference genes gave hints to an increased gene expression in donors who died of a hypoxia associated cause of death compared to the control group. Moreover, this study confirmed the particular importance of the elected normalisation strategy. Surprisingly, the four genes of interest suggested a decrease in gene expression after its normalisation against GAPDH as a single nonvalidated control gene. A possible reason for this discrepancy may be found in the apparent instability and the co-regulation of GAPDH under hypoxic conditions. To summarize, the presentation of validated control genes as well as the analysis of parameters affecting RNA integrity and quantitative gene expression data provide a basis for the reliable performance of future gene expression studies using human autopsy samples. This raises hope for the successful implementation of postmortem gene expression studies as additional tool in forensic pathology to assist the determination of somebody´s cause of death

Multidrug resistance (MDR) in cancer is characterized by cross-resistance to a variety of structurally unrelated chemotherapeutic compounds. Altered expression levels of P-glycoprotein (Pgp) and multidrug resistance-associated protein-1 (MRP1) are strongly associated with MDR. Pgp and MRP1 are members of the ATP-binding cassette (ABC) superfamily of membrane proteins. Members of this family transport a wide spectrum of substrates (e.g. peptides, metals, ions, toxins and chemotherapeutic drugs) across cell membranes. Completion of the human genome-sequencing project revealed there are close to 50 ABC genes (Dean et al., 2001); however, function or substrate specificity is known for only a few. It is anticipated that several of these proteins may also contribute to MDR and mutations or altered expression levels of a number of ABC proteins are causal of diseases such as cystic fibrosis and Tangier disease. Despite these associations, there has been no systematic attempt to profile expression of ABC transcripts. We have developed a competitive-RT-PCR assay to examine RNA expression profiles of 35 human ABC transcripts. This assay uses RNA competitors for each transcript, and fluorescently labeled PCR products are resolved using capillary electrophoresis. The data is highly reproducible and accurate. We have examined ABC transcript expression profiles in sensitive and vincristineresistant cell lines, as well as cells exposed short-term (24 hours) to various chemotherapeutic drugs. Vincristine-selection demonstrated multiple, possibly sequential, expression changes may collectively contribute to MDR, whereas short-term drug exposure revealed few expression changes, suggesting that altered mRNA expression is not a common response to xenobiotic exposure. ABC expression profiles were also generated for 12 normal human tissues. Analysis of individual transcripts and the collective expression profiles provided insights into the relative contribution of ABC proteins to MDR, defense against xenobiotic toxins and phospholipid and cholesterol homeostasis. Another study examined the effect o f enforced expression o f the proapoptotic BAX cDNA on the collective ABC expression profile and relative drug-resistance of a cell line. Our results suggest Pgp-mediated MDR may be regulated by BAX protein in some cell lines. The continued systematic evaluation of ABC transcript profiles under various experimental conditions should provide further insights into the normal physiological roles of ABC proteins.

RNA molecules isolated from FFPE samples are highly fragmented and modified, and generally deemed unsuitable for downstream gene expression profiling. With the development of molecular biology, there has been growing interest in profiling archival FFPE samples. Successful profiling of transcripts from FFPE samples would greatly expand tissue sources for large scale gene expression studies; also it would pave the way for future applications on the type of tissue readily available in the clinical setting. So far, there is a lack of systemic studies evaluating the quality of RNA isolated from routinely processed FFPE samples, and it has remained difficult to assess how well FFPE-derived RNA mirrors the status of RNA isolated before fixation. In this project, the similarity of miRNA and mRNA profiles between matched frozen and FFPE lymphoid hyperplasia tissues (N=7 for miRNA comparison, N=4 for mRNA comparison) were evaluated. We found consistently good correlation (mean of Pearson coefficient=0.939, mean of Spearman coefficient=0.905, mean of Kendall tau=0.744) between matched frozen and FFPE-derived miRNA profiles, suggesting FFPE samples may retain miRNA expression information quite well. This has major positive implications for research using FFPE samples, as miRNA profiling becomes more prominent in bioprofiling studies. On the contrary, mRNA isolated from FFPE samples showed less correlation (Spearman coefficient less than 0.75) with its frozen counterpart on the Agilent microarray platform. With a post extraction heat treatment aimed at reversing base modifications and cross linking structures, obvious global mRNA quality improvement was observed in cases where samples appeared to be heavily cross linked, but was less effective and even detrimental in cases where cross linking was less prominent. This research suggests that the extent of cross linking may be critical in terms of determining whether a particular FFPE tissue will become a useful source of mRNA for global profiling studies<br>Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-26 10:49:50.044

Les protéines sont responsables de pratiquement toutes les fonctions performées au sein du corps cellulaire et de ses alentours. Le contrôle de l’expression génique détermine l’abondance, la localisation et le moment de la production de protéines dans la cellule. Il s’agit de l’un des processus centraux à la régulation de la physiologie et du fonctionnement cellulaire. La moindre perte de balance dans ce complexe système engendre des conséquences majeures sur l’intégrité cellulaire, menant au développement de plusieurs maladies parfois incurables. La traduction de l’ARN messager en produit protéique constitue la dernière étape de l’expression génique. Elle est régulée de plusieurs façons, intrinsèques et extrinsèques à la séquence. Il s’agit également du processus cellulaire le plus coûteux en termes d’énergie. Le profilage des ribosomes (Ribo-Seq) figure parmi les récentes et prometteuses technologies ayant permis une meilleure étude des mécanismes de régulation de la traduction. Ces résultats contiennent toutefois la présence de variabilité et de bruits de nature infondée. Ce travail présente la mise en place d’une stratégie permettant la dissociation de signaux d’origine biologique de ceux ayant une origine technique. Ceci est effectué au travers de la mise en place de profiles consensus de densité ribosomale extrait d’une analyse comparative de plusieurs expériences de Ribo-Seq chez la levure (Saccharomyces cerevisiae). Les signaux biologiques dérivés par les profils consensus correspondent avec les signatures de pauses ribosomales connues, telles que les scores de repliements de l’ARNm et la charge des acides aminés. Épatamment, notre stratégie a également permis l’identification de séquences différentiellement transcrites (DT). Ces dernières jouent un rôle sur la cinétique de la phase d’élongation de la traduction, elles comportent notamment une surreprésentation de codons associés aux modifications des ARNs de transfert (tRNAs). Elles se retrouvent d’ailleurs impliquées dans le maintien de l’homéostase cellulaire, ayant une présence marquée chez des gènes prenants part aux mécanismes de biosynthèse de la macromolécule ribosomale ainsi que chez les ARNms aux sublocalisations cellulaires précises, notamment chez les mitochondries et le réticulum endoplasmique (ER). En plus de démontrer les possibilités de découvertes offertes par la technique du Ribo-Seq, cette étude présente une évidence de la nature dynamique et hétérogène du processus de traduction chez la cellule eucaryote. Elle démontre également le rôle de l’information directement encodée dans la séquence dans l’optimisation générale de l’homéostasie cellulaire.<br>Proteins are responsible for virtually all functions performed within and in the surroundings of a cell. The control of gene expression, which determines the amount, localisation and timing of protein production in the cell, is the central processes in the regulation of cellular physiology and function. Any disturbance in this complex system can generate important consequences on cellular integrity, sometimes leading to incurable diseases. The translation of messenger RNA into a protein product is the last step of the gene expression mechanism. It can be regulated in manifold ways, both intrinsically and extrinsically to the transcript sequence. It is also the costliest cellular process in terms of energy. Ribosome profiling (Ribo-Seq) is one of the recent and promising technologies making it possible to better study the mechanisms of translation regulation. Its results have however been shown to display variability in reproducibility and to contain noise of uncharted sources. This work presents the implementation of a strategy for dissociating signals of biological origin from those of technical origin. This is performed by the computation of a consensus profile of ribosomal density derived from a comparative analysis of several Ribo-Seq experiments in yeast (Saccharomyces cerevisiae). The biological signals derived by the consensus profiles correspond with signatures of known ribosomal pauses, such as mRNA folding strength and amino acid charges. Amazingly, our strategy also enabled the identification of differentially transcribed (DT) sequences. The latter have shown an over-representation of codons associated with modifications of transfer RNAs (tRNAs). They are also involved in the control of cellular homeostasis, exhibiting a marked presence in genes involved in ribosome biosynthesis as well as in mRNAs with precise translation sub-localization, particularly in mitochondria and the endoplasmic reticulum (ER). In addition to demonstrating the possibilities of discovery offered by the Ribo-Seq technique, this study also presents evidence of the dynamic and heterogeneous nature of the translation process in the eukaryotic cell. It also showcases its diverse regulatory mechanisms and the role of information directly encoded in the sequence in the general optimization of cellular homeostasis.

The presence of introns in all the eukaryotic genomes identified so far underscores the fundamental and ubiquitous role of pre-mRNA splicing. The spliceosomal machinery, comprised of five small nuclear RNAs and several protein factors, catalyzes the two-transesterification reactions of splicing with precision and consistency. Through a complex network of protein-protein and RNA-protein interactions it ensures the removal of the intron and ligation of the flanking exons to yield the mature mRNA. Prpl7 is a splicing factor that functions at the second-step of splicing (Vijayraghavan et all, 1989). Null alleles of prpl7 are viable at 23°C but die at temperatures above 33°C (Jones et al.9 1995). Besides its functions in pre-mRNA splicing, mutants in PRP17ICDC40 were independently shown to affect cell-cycle progression, particularly the Gl/S and G2/M transitions (Chawla et a/., 2003). In this study, we have attempted a further characterization of Prpl7 to analyze both its role in pre-mRNA splicing and in cell-cycle progression with an aim to decipher underlying reasons for the interlinking of these two cellular processes. Different experimental approaches were adopted to achieve this goal. Global gene-expression profiling provided an overview of all the transcripts affected in a prpl 7 mutant and allowed its comparison with mutants of other splicing factors. This exercise aided in identification of both pre-mRNA splicing and cell-cycle related effects of Prpl7. Biochemical analysis of the Prpl7 spliceosomal associations have provided further clarity on the part played by Prpl7 in pre-mRNA splicing. A genome-wide two-Hybrid screen for interacting partners of Prpl7 was undertaken and uncovered two Likely interacting partners of Prpl7. Global expression profiling of splicing mutants Pleiotropic phenotypes observed in mutants of prpl 7 and few other splicing factors have been speculated to arise from either the multi functionality of the factor or more likely due to a specific requirement of the factor in splicing of a select subset of transcripts, that encode proteins essential to the affected cellular pathway. These observations raise questions about the ubiquitous requirement of factors in pre-mRNA splicing. To understand these aspects of splicing, we studied the effects of splicing factor mutants on a genome-wide scale. Using splicing-sensitive DNA microarrays imprinted with all yeast ORFs and in addition, independent spots for a majority of the intron sequences, we analyzed the global expression changes triggered by the inactivation of temperature-sensitive mutations in PRP17 or PRP22. Experiments with prp2-l mutant strain detect, as expected, an increase in pre-mRNA levels at the intron spots and further demonstrated that the ORF spots detect a decrease in mRNA levels in these DNA microarrays. These results established the DNA micro arrays as tools for the analysis of splicing on a global scale. The temporal alterations in transcript profiles in prpl 7 and prp22 mutants, as compared to the wild type, revealed both shared and unique effects of these factors on clusters of intron-containing transcripts. Such differential effects, on intron-containing transcripts, amongst the splicing mutants implicate specialized roles for each of these factors. Through analysis of the set of intron-containing transcripts affected in prpl7Δ cells, we infer those attributes of these pre-mRNA substrates, which predispose a need for Prpl 7. We find that splicing of introns longer than 200nts has a stronger dependence on Prpl7. The distance between consensus intron elements- the branch-nucleotide and the 3'splice-site (B), also imposes a requirement for Prpl7. Introns with a 13nts or lesser distance between these elements are spliced even in the absence of Prpl 7, both in vivo and in vitro. The 5'splice-site to branch-nucleotide distance (A) also influences the need for Prpl7. Most introns with a A/B ratio of less than 2 undergo Prpl7 independent splicing in vivo. Intron-containing genes that could be responsible for the pleiotropic phenotypes of prpl7 were also identified through the global splicing analysis. These included splicing targets that act at the Gl-S phase such as ANC1/TAF14, TMD4, PHO85 and those at the G2-M phase of the cell-cycle; TUB], TUB3, GIM5, MOBl UBC9. Recently, a different study implicates ANC1ITAF14 as the intron-containing gene responsible for the cell-cycle phenotype associated with prpl7 (Dahan and Kupiec, 2004). Our global analysis of all intron-containing transcripts with compromised expression in prpl7A cells identify, in addition, PHO85 as a possible regulator underlying cell-cycle effects in this mutant. Pho85 is a cyclin-dependent kinase that functions at both the Gl/S and M/Gl phases of the division cycle (Moffate* al., 2000). Synergistic growth defects in double mutants of prpl7 and pho85 have uncovered a novel role for Prpl7 in bud morphogenesis. Our micro array data also reveals compromised expression levels for several key intronless cell-cycle rregulatory genes indicating a possible splicing-independent role for Prpl7 in the cell-cycle. Examples of such transcripts are: the Gl cyclins CLN1, CLN2 and CLN3; CDC6, required for assembly of the pre-replication complex at sites of replication origin; and the cell-cycle regulatory transcription factors: SWI5 and ACE2. The global analysis has therefore enabled, for the first time, a characterization of the splicing substrate specificity of Prpl7 and has also uncovered the effects of this protein on gene expression during cell-cycle progression (Fig. V.I A). Spliceosomal interactions of Prpl7 To understand the function and associations of Prpl7 in the spliceosome, we have examined its snRNP interactions and determined the time point of its coalescence on assembling spliceosomes. A functional epitope tagged-Prpl7 was created using the polyoma middle T-antigen and the poly-HIS tags (Stevens et aln 1999). Through immunoprecipitation analyses performed with splicing extracts, from this strain, we find Prpl7 to associate with three spliceosomal snRNPs- U2, U5 and U6, implicating an interaction with active spliceosomes or post-splicing complexes. Specific biochemical depletion of any one of these snRNAs, through oligo-directed RNaseH cleavage, did not have a drastic effect on the association of Prpl7 with the other two snRNAs. To decipher the point at which Prp 17 joins the assembling spliceosomes, we examined the presence of Prp 17 in in vitro assembled complexes generated under various conditions. The conditions adopted were designed to stall and enrich for •assembly intermediates. A co-immunoprecipitation of the input precursor RNA and reaction intermediates revealed an early association of Prp 17 with the assembling Spliceosome prior to its catalytic activation. This association occurred in the A2-1 complex, which contains the U4/U6.U5 tri-snRNP along with the Ul and U2snRNPs. Prpl7 was found to associate with all subsequent complexes until the completion of catalytic transesterification reactions and possibly continue with the spliced-out introns complex (Fig. V.1B). Identification of two novel interacting partners of Prpl7 from a genome-wide two-hybrid screen Although several genetic interacting partners of PRP17 are known, none display a direct physical association with Prpl7. Knowledge of the proteins that Prpl7 interacts with can further the functional characterization of this protein and aid in deciphering its link to cell-cycle progression. A genome-wide screen for interacting partners using Prpl7 as bait was carried out in a two-hybrid system with a yeast genomic DNA-B42 activation-domain library (Gyuris et al., 1993). Through this screen we identified two interacting partners of Prpl 7- YOL078W, an essential gene and SGML The domain in the 1176 amino acid YOL078W protein responsible for interaction with Prpl7 was mapped to a 225 amino acid segment in the C-terminai region of this protein. The N-terminal region of the protein appears to exert a negative effect on the interaction with Prpl7. While YOL078w does not have any apparent role in pre-mRNA splicing, a majority of the cells arrest with small buds indicating a late Gl or early S phase arrest upon transcriptional shut-down of YOL078W. YOL078W has been independently characterized as AVOl, a component of the TOR complex, involved in nutrient sensing and cell size regulation (Loewith et al, 2002). Other reports show it tto be a component of a complex that interacts with Ceglp, a nuclear protein involved in mRNA capping (Gavin et al, 2002). We hypothesize that Prpl7 and Avol may exist in a dynamic nucleocytoplasmic complex possibly functioning in either cell-cycle regulation, RNA processing or both. Through this study we have Established the use of splicing-sensitive microarrays as tools for the characterization of pre-mRNA splicing factors. Simultaneous assessment of the effects on other cellular pathways was accomplished through expression profiling of all the intron-containing and intronless genes. Deciphered the differential dependence of pre-mRNA substrates on spliceosome factors at a global scale. Predicted the substrate-specificity of the second-step splicing factor, Prpl7, and verified some of these predictions in vitro. Gathered evidence for a possible splicing-independent effect of Prpl7 on the cell division cycle. Uncovered a novel function of Prpl7 in bud morphogenesis, as deduced from its synergistic genetic interaction with PHO85. Identified U2, U5 and U6 snRNPs as interacting partners of Prpl7 in both xtracts and in in vitro splicing reactions. Determined the point of coalescence of Prpl7 during spliceosome assembly to be at an early assembly stage soon after the entry of U4/U6.U5 tri-snRNP and prior to catalytic activation. Demonstrated continued Prpl7 association with the spliceosome beyond the completion of the splicing reactions. Identified Avolp and Sgmlp as novel interacting partners of Prpl7 through a genome-wide two-hybrid screen.

碩士<br>高雄醫學大學<br>醫藥暨應用化學研究所<br>96<br>Human embryonic stem (hES) cells have the capacities to be propagated for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. Previous reports have shown that maintenance of undifferentiated hES cells requires culturing on mouse embryonic fibroblast (MEF) feeders. However, this culture system contains animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hES cells. Besides, the molecular mechanisms underlying pluripotency are not yet fully understood. Here a feeder-free culture system was developed and the expression profilings of mRNAs and mircoRNAs of hES cells cultured in different feeder-free conditions were compared with those cultured on MEF feeder. The hES-T3 cells with normal female karyotype cultured on MEF feeder (T3MF) and on Matrigel in MEF-conditioned medium (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on either MEF in hES medium (containing 4 ng/ml bFGF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF). However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in defined medium supplemented with 4 ng/ml bFGF and 5 ng/ml Activin A (T3BA) was found to be different from those of T3MF and T3CM cells. In T3BA cells, seven microRNAs (four hES cell-specific microRNAs miR-372, 302d, 367, 200c and three other microRNAs miR-199a, 19a and 217) were found to be up-regulated, whereas five miRNAs (miR-19b, 221, 222, let-7b and let-7c) were down-regulated by Activin A. Furthermore, seven abundantly (more than 3-fold of overall mean) differentially (more than 3-folds of changes) expressed mRNAs of NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, COX6A1 and CD24 genes targeted by miR-372, 302d, 367, 200c, 199a, 19a and/or 217 were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3MF, T3CM and T3BA cells. The abundantly expressed mRNAs in T3MF, T3CM and T3BA cells were also analyzed for the network and signaling pathways, and roles of Activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the hES cell phenotype and function.