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Translation and mRNA degradation are tightly regulated upon stress where protein synthesis and mRNA decay are modulated to optimize the stress response. However, the mechanisms that regulate mRNA decay and translation during stress are not fully understood. In this thesis, I show that Dcp2, a major decapping enzyme, undergoes phosphorylation by Ste20 kinase during stress and promotes stabilization of ribosomal protein mRNAs as well as Dcp2 accumulation in Processing bodies (P-bodies) in Saccharomyces cerevisiae. In addition, I have analyzed the role of P-bodies by examining how alterations in P-body assembly factors affect the transcriptome. Interestingly, I observe that Edc3, a component of P-bodies that promotes their assembly, can either stabilize or destabilize specific subsets of yeast mRNAs. I also show that Lsm4, a P-body component that mediates the assembly of P-bodies along with Edc3, promotes mRNA decay via its aggregation domain. These results argue that P-bodies can function as sites of mRNA degradation and storage for a subset of mRNAs by the localized accumulation of specific factors.

No description available.

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Insulin-like growth factor 1 (IGF-1) acts as a mediator for several hormones in bone. In rat the IGF-1 gene is processed into distinct mRNA molecules which differ in their 5' and 3' ends. Two major classes of mRNA transcript are produced which contain either exon 1 (Class 1) or exon 2 (Class 2). The main aims of this study were to identify the IGF-1 splice variants produced in bone and investigate possible effects of GH on transcript expression. IGF-1 transcripts were analysed in bone using reverse transcription polymerase chain reaction (RT-PCR) using forward specific primers for exon 1 and exon 2 and reverse primers for exon 5 and 6. Analysis of RNA extracted from either whole tibia, growth plate, primary osteoblasts, and clonal cell lines UMR 106 and ROS 17.6 showed distinct patterns of expression. All IGF-1 splice variants were expressed in bone tissue and primary osteoblasts however none of the variants were detected in UMR 106 cell line and only Class 1 Ea was detected in ROS 17.6 cell line. No obvious change in this pattern of expression was observed on GH stimulation of these primary cells and osteosarcoma cell lines. However, GH was found to stimulate osteoblast growth by enhancing proliferation of a subpopulation of cells but GH did not effect alkaline phosphatase production. GH did not increase total IGF-1 mRNA levels in osteoblasts as detected using RNase Protection assay but there was a change in the splicing profile such that Class 2 transcripts were increased by 150% (p=0.05) and Class 1 transcripts decreased by 35% (p=0.02). Furthermore antisense ODNs directed against the common exon 4 ofIGF-1 caused a dramatic increase in osteoblast apoptosis whereas sense and scrambled control ODNs had no effect. Our data show that IGF-1 is an important constitutively expressed factor in osteoblasts and GH may exert its actions by increasing Class 2 transcripts in bone which could have paracrine effects on neighbouring cells.

Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-30Bitstream added on 2014-06-13T20:41:05Z : No. of bitstreams: 1 apponi_lh_dr_araiq.pdf: 2630425 bytes, checksum: bd39235826e1f6c4a56f84fffab81fff (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A expressão gênica pode ser regulada em eucariotos em diversas etapas dometabolismo de mRNA, como transcrição, processamento, tradução e degradação. A estabilidade de mRNA é modulada por elementos presentes no transcrito e por proteínas ligantes de RNA associadas a esses elementos. Pub1 de S. cerevisiae é uma proteína citoplasmática capaz de estabilizar transcritos contendo elementos ricos em AU (ARE e ARE-like) ou elementos estabilizadores (STE). O presente trabalho identificou num rastreamento de duplo-híbrido a proteína Nab2 como ligante de Pub1. Nab2 é uma proteína nucleocitoplasmática essencial que regula o comprimento da cauda poli(A) e a exportação nuclear de mRNA. A interação entre Pub1 e Nab2 foi confirmada por co-purificação e ensaio de interação in vitro. Foi demonstrado também que essa interação é mediada pelo domínio de dedos de zinco presente na região C-terminal de Nab2. A análise da relação funcional entre essas duas proteínas revelou que Nab2, assim como Pub1, é capaz de modular a estabilidade de mRNA. A estabilidade do transcrito de RPS16B, mensageiro contendo sequência ARE-like e regulado por Pub1, é diminuída nos mutantes nab2- 1 e nab2-67. No entanto, a estabilidade do transcrito de GCN4, mensageiro contendo STE e também regulado por Pub1, não é afetada nos mesmos mutantes. Resultados semelhantes foram observados para outros transcritos contendo sequências ARE-like ou STE. Ainda, dados obtidos com um mutante da via NMD (?upf1) mostraram que esta via de decaimento não está envolvida com o mecanismo de estabilização de RPS16B mediada por Pub1 e Nab2. Uma análise mais profunda mostrou que a sequência ARE-like presente no mensageiro de RPS16B é necessária para a estabilização mediada por Nab2. A proteína Pub1 e seus domínios isolados foram produzidos e purificados, mas não foi possível a obtenção de cristais para... / Regulation of gene expression can occur at different levels of mRNA life cycle, including transcription, processing, translation and degradation. mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA-binding proteins. Poly(U)-binding protein 1 (Pub1) is a cytoplasmic S. cerevisiae mRNA binding protein that stabilizes transcripts containing AU-Rich Elements (ARE and ARE-like) or Stabilizer Elements (STE). In a yeast two-hybrid screen, we identified Nuclear poly(A)-binding protein 2 (Nab2) as a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by co-purification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific target mRNA transcripts. We find that the half-life of the RPS16B transcript, an ARE-likecontaining Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained with other ARE- and STE-containing Pub1 target transcripts. Additionally, results obtained with a mutant of the NMD pathway (?upf1) showed that this pathway is not involved in the mechanism of RPS16B stabilization mediated by Pub1 and Nab2. Further analysis reveals that the ARE-like sequence is necessary for Nab2- mediated transcript stabilization. Full-length Pub1 and isolated domains were produced and purified, however, it was not possible to obtain protein crystals for tertiary structure determination. Taken together, these results suggest that Nab2 acts together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events involved in mRNA biogenesis...(Complete abstract click electronic access below)

The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.

The strategy of producing expressed sequence tags (ESTs) is greatly used for gene mining and analysis of gene expression. An EST is a partial and single-pass sequence generated from a complementary DNA (cDNA) library by random selection, and is typically about 400 to 600 bases. Here, we constructed and analyzed a brain EST library from 10-day-old (10-day posthatching) tilapia, Oreochromis mossambicus, to assist in investigating the relation between brain neural development and neuroendocrine and brain sexual differentiation during the critical period of sexual dimorphism by understanding brain gene expression, and, furthermore, to find genes that are important in brain but are unknown nowadays. A total 1,124 ESTs were in the 10-day-old tilapia brain EST library, and 1,092 ones were readable, and after discarding one that is too short (<200bp) or is totally vector sequence from the readable ESTs, 1,084 ESTs were assembled and then analyzed. 108 contigs from 261 ESTs, 20 kinds of similar sequences from 40 ESTs, and 783 singletons from the 1,084 ESTs were found, and after aligning in turn with the non-redundant (nr) protein database, nr nucleotide database and EST database dbEST supported by the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) respectively, the results are as follows: 57 and 16 contigs including 146 and 37 ESTs matched with sequences in the nr protein and nr nucleotide databases respectively, and the rest 35 contigs including 78 ESTs were novel sequences; nine, one and two kinds of the similar sequences including 18, two and four ESTs matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest eight kinds of the similar sequences including 16 ESTs were novel sequences; 309, 52 and 47 singletons matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest 375 singletons were novel sequences. As a whole, the most functions of proteins that the contigs, similar kinds of sequences, and singletons from the 10-day-old tilapia brain EST library matched with were binding and transport activity. Recently, many researchers provide reasonable explanations for evolution, relation between genomic polymorphism and drug effects, and isoforms presentation of known proteins by collecting ESTs from open EST databases or from EST libraries they constructed, and we believe that this 10-day-old tilapia brain EST library can promote our ability to resolve questions about the topic we are researching in.

The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.