<p>RPL23a is one of the ~80 ribosomal proteins (r-proteins) of the cytoplasmic ribosome in the model plant <i>Arabidopsis thaliana</i>. The objectives of this research were to establish Arabidopsis RPL23a as a functional r-protein, characterize expression patterns for the two genes (RPL23aA and B) encoding RPL23a using reverse transcription PCR (RT-PCR), and identify regulatory elements controlling the expression of RPL23aA and B. Complementation of a yeast l25 mutant with AtRPL23aA demonstrated that AtRPL23aA can fulfill all the essential functions of L25 in vivo. A survey of various Arabidopsis tissue types showed that, while RPL23aA and B expression patterns both showed increased transcript abundance in mitotically active tissues, RPL23aB transcript levels were generally lower than those of RPL23aA and responded differently to abiotic stresses. In order to determine cis regulatory elements controlling RPL23aA and B expression, the 5 regulatory region (RR) of each gene was characterized via plants carrying a series of 5 RR deletion fragments upstream of a reporter. Transcript start sites and 5 untranslated regions (UTRs) for both RPL23aA and B were also characterized using primer extension, and transcripts from 5 deletion transgenics were amplified using RT-PCR. No correlation was observed between putative cis-acting elements and the expression patterns from the RPL23aA and B deletion transgenics, although a 102 bp sequence in the RPL23aB 5 RR was found to contain pollen-specific elements. 5 leader introns were found in each RPL23a gene, and amplification of transgene transcripts from deletion series plants indicated the importance of post-transcriptional and translational regulation in RPL23aA and B expression. This thesis work is the first demonstration of a plant RPL23a protein as a functional member of the L23/L25 (L23p) conserved r-protein family, and is one of the few in-depth studies of the regulation of r-protein genes in plants. While the majority of previous research on plant r-protein gene expression has focused solely on transcript levels, I show herein that post-transcriptional mechanisms have a critical role in regulating these genes, and thus plant r-protein genes more strongly resemble their mammalian counterparts than those of yeast in terms of structure and regulation.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal protein genes are crucial for ribosome biogenesis. The ribosome itself is responsible for protein synthesis and hence cellular growth and development. Intertwining network of proteins in conjugation with cellular environment such as nutrition and growth factors collectively regulate expression of the ribosomal protein genes. DNA microarray analysis has implicated the role of 26S proteasome in transcriptional regulation of the ribosomal protein genes tying protein degradation to protein synthesis pathway. To determine the mechanism as to how the 26S proteasome promotes transcription of the ribosomal protein genes a series of experiments were performed. The results reveal that the 19S subcomplex of the 26S proteasome is recruited to the promoters of the ribosomal protein genes in a TOR (Target of Rapamycin)-dependent manner. TOR signals environmental cues and controls the expression of the ribosomal protein genes. Thus recruited 19S proteasome subcomplex promotes transcriptional initiation via facilitation of the recruitment of co-activator NuA4 (Nucleasome acetyltransferase of histone H4) complex to activator Rap1. NuA4 enhances PIC (Pre-initiation complex) formation at the core promoter, but it is not clearly understood how does it do so. Researches have identified two different forms of TBP: TAF (TBP associated factor)-dependent form of TBP and TAF-independent form of TBP. This work shows that impaired association of NuA4 interferes with TFIID recruitment, but recruits TAF-independent form of TBP to the core promoter. This recruitment of TBP is dependent on SAGA (Spt-Ada-Gcn5-acetyltransferase). Like ribosomal protein genes, antisense transcription is also enhanced by TAFs. However, it remains unknown whether NuA4 also promotes TAF-regulated antisense transcription. The results illustrate that like ribosomal protein genes, transcription of GAL10 antisense is also promoted by NuA4 HAT (histone acetyl transferase). NuA4 HAT is recruited to the 3’-end of the GAL10 coding sequence, acetylates histone H4 and promotes GAL10 antisense transcription. This work also reveals the roles of other chromatin regulatory factors in controlling antisense transcription. Collectively, these results significantly advance our current understanding of the regulatory mechanisms of ribosomal protein genes’ expression and antisense transcription. The ribosome and antisense are involved in virtually all the biological processes. Aberrant expression of the ribosomal protein genes and antisense transcripts are associated with numerous human disorders including cancers and cardiovascular diseases. Therefore, analyses of their regulatory processes provide valuable information toward understanding the etiology of numerous human diseases with potential therapeutic interventions.

A temperature-sensitive, protein synthesis-defective mutant of Escherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to the rplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.

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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

A proteína ribossomal L10 (RP L10) é uma forte candidata a ser incluída na classe de proteínas supressoras de tumor. Também denominada QM, a proteína em questão é conhecida por participar da ligação das subunidades ribossomais 60S e 40S e da tradução de mRNAs. Possui massa molecular entre 24 a 26 kDa e ponto isoelétrico (pI) 10,5. A seqüência da proteína QM é bastante conservada em mamíferos, plantas, invertebrados, insetos e leveduras indicando que esta possui funções críticas na célula. Com função supressora de tumor, a proteína RP L10 foi estudada em linhagens de tumor de Wilm (WT-1) e em células tumorais de estômago, nas quais se observou uma diminuição na quantidade de seu mRNA. Mais recentemente a RP L10 foi encontrada em baixas quantidades nos estágios iniciais de adenoma de próstata e com uma mutação em câncer de ovário, indicando uma participação no desenvolvimento destas doenças. Como proteína, já foi descrito que esta interage com as proteínas c-Jun e c-Yes, inibindo a ação ativadora de fatores de crescimento e divisão celular. Este trabalho tem um papel importante no estabelecimento da expressão desta proteína solúvel, para estudos posteriores que tenham como objetivo avaliar a ação de regiões específicas que atuam na ligação das subunidades ribossomais 60S e 40S e tradução, bem como nas regiões que se ligam a proto-oncogenes. O cDNA para proteína QM foi amplificado por PCR e clonado no vetor de expressão periplásmica p3SN8. A proteína QM foi expressa em E.coli BL21 (DE3) no citoplasma e periplasma bacteriano e na melhor condição, a expressão de QM de bactérias transformadas pelo plasmídeo recombinante p1813_QM em 25°C ou 30°C, a proteína foi obtida solúvel e com quantidad es muito pequenas de contaminantes. Os ensaios de estrutura secundária demonstraram que a proteína QM tem predominância de a-hélice, mas quando do seu desenovelmento, essa condição muda e a proteína passa a ter característica de folhas &beta;. / The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm\'s tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribossomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813_QM at 25°C or 30°C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by &beta;- sheet feature.

A proteína ribossomal L10 (RP L10) é uma forte candidata a ser incluída na classe de proteínas supressoras de tumor. Também denominada QM, a proteína em questão é conhecida por participar da ligação das subunidades ribossomais 60S e 40S e da tradução de mRNAs. Possui massa molecular entre 24 a 26 kDa e ponto isoelétrico (pI) 10,5. A seqüência da proteína QM é bastante conservada em mamíferos, plantas, invertebrados, insetos e leveduras indicando que esta possui funções críticas na célula. Com função supressora de tumor, a proteína RP L10 foi estudada em linhagens de tumor de Wilm (WT-1) e em células tumorais de estômago, nas quais se observou uma diminuição na quantidade de seu mRNA. Mais recentemente a RP L10 foi encontrada em baixas quantidades nos estágios iniciais de adenoma de próstata e com uma mutação em câncer de ovário, indicando uma participação no desenvolvimento destas doenças. Como proteína, já foi descrito que esta interage com as proteínas c-Jun e c-Yes, inibindo a ação ativadora de fatores de crescimento e divisão celular. Este trabalho tem um papel importante no estabelecimento da expressão desta proteína solúvel, para estudos posteriores que tenham como objetivo avaliar a ação de regiões específicas que atuam na ligação das subunidades ribossomais 60S e 40S e tradução, bem como nas regiões que se ligam a proto-oncogenes. O cDNA para proteína QM foi amplificado por PCR e clonado no vetor de expressão periplásmica p3SN8. A proteína QM foi expressa em E.coli BL21 (DE3) no citoplasma e periplasma bacteriano e na melhor condição, a expressão de QM de bactérias transformadas pelo plasmídeo recombinante p1813_QM em 25°C ou 30°C, a proteína foi obtida solúvel e com quantidad es muito pequenas de contaminantes. Os ensaios de estrutura secundária demonstraram que a proteína QM tem predominância de a-hélice, mas quando do seu desenovelmento, essa condição muda e a proteína passa a ter característica de folhas &beta;. / The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm\'s tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribossomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813_QM at 25°C or 30°C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by &beta;- sheet feature.