Publicação
Pabpn1 protein : aggregation properties in an opmd cellular model and a novel role in splicing
| Resumo: | Eukaryotic gene expression is a complex stepwise process that begins with transcription initiation and terminates with the synthesis of a specific protein. Before being transported out of the nucleus as mRNAs, primary gene transcripts undergo a series of co- and post-transcriptional maturation events. During transcription, the nascent transcript is capped in its 5´ end, introns are spliced and the 3´end is cleaved and polyadenylated. The mature mRNA is then released from the site of transcription and exported to the cytoplasm for translation. Although these transcriptional maturation processes tend to be studied in a rather simplistic isolated manner, ample evidence exists demonstrating that the various events that constitute pre-mRNA maturation occur in an integrated fashion. Distinct machines carry out each of the steps in the gene expression pathway but despite the unique reactions they catalyse, each machine also interfaces both physically and functionally with other machines in the pathway. More specifically, a wide number of studies have highlighted the role of the Carboxyl Terminal Domain of the largest subunit of RNA Polymerase II as the key determinant player in the coupling of the pre-mRNA maturation events. In this work we have addressed the question of the co-ordination of all the steps of pre-mRNA processing using the poly(A) binding protein nuclear 1 (PABPN1) as a tool. PABPN1 is an abundant nuclear protein that stimulates poly(A) polymerase (PAP) during polyadenylation of mRNA precursors in metazoan cells. PABPN1 acts primarily as a stimulatory factor for the extension of the poly(A) tail and remains associated to the mRNA after the poly(A) addition, travelling with the mRNA to the cytoplasm where translation occurs. In addition to the very well characterized role in stimulating PAP, there are some other reports of additional PABPN1 functions. For instance, PABPN1 has been found in the nuclear nonsense-mediated decay complex, which is thought to complete a "pioneer" round of translation checking the integrity of the mRNA. PABPN1 has also been shown to associate with RNA Polymerase II along the chromatin axis and has been proposed to participate in transcription by associating with the Ski-interacting pre-spliceosomes, thus likely to be recruited protein and with MyoD. It has also been identified as a component of the spliceosome, neither stably associated with U4/U6.U5 tri-snRNPs nor present in purified to the spliceosome at a later, more active, stage of its assembly. Our results demonstrate that PABPN1 plays an unforeseen role in the splicing reaction. Combining a series of in vitro pulldown and immunoprecipitation assays, PABPN1 is shown to interact directly with very well characterized splicing factors and to be recruited to intronic sequences upon gene transcription activation. PABPN1 is also shown to modulate splice site choices in splicing reporter mini-genes, therefore implicating PABPN1, directly or indirectly, in alternative splicing regulation. In addition to its novel role in splicing, we also tried to understand the role of PABPN1 in a human genetic disease called Oculopharyngeal Muscular Dystrophy (OPMD). In 1998, it was found that mutations on the PABPN1 gene were responsible for OPMD. The mutation consists of short (GCG) expansions that result in the expansion of a polyalanine stretch at the N-terminus of the protein. Following the initial methionine, the wild type PABPN1 protein contains a naturally occurring trinucleotide repeat (GCG)6 coding for the first 6 alanines in a 10 tandem alanine stretch. In the OPMD patients, the (GCG)6 repeat is expanded to (GCG)8-13, thus leading to an expansion of the polyalanine tract from 10 to 12-17 alanines in the N-terminus of PABPN1. Because intranuclear inclusions containing PABPN1 represent a pathological hallmark of muscle cells from OPMD patients, it has been suggested that the mutated PABPN1 induces or facilitates the formation of the inclusions. To investigate whether this was the case, we have generated green fluorescent protein (GFP) fusions with PABPN1 variants. These fusion proteins were transiently expressed in human HeLa cells and in the murine myogenic cell line C2. We showed that, upon over-expression, PABPN1 aggregation can occur in the absence of polyalanine expansion. We further demonstrated that propensity of PABPN1 to form intranuclear inclusions is coupled to stimulation of polyadenylation, and protein aggregation is a highly dynamic, reversible process. Our results contributed greatly for the interpretation of OPMD model systems based on exogenous expression of PABPN1. |
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| Autores principais: | JP |
| Assunto: | Proteína PABPN1 Agregação celular Splicing alternativo Poli A União de RNA Teses de doutoramento - 2007 |
| Ano: | 2007 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Eukaryotic gene expression is a complex stepwise process that begins with transcription initiation and terminates with the synthesis of a specific protein. Before being transported out of the nucleus as mRNAs, primary gene transcripts undergo a series of co- and post-transcriptional maturation events. During transcription, the nascent transcript is capped in its 5´ end, introns are spliced and the 3´end is cleaved and polyadenylated. The mature mRNA is then released from the site of transcription and exported to the cytoplasm for translation. Although these transcriptional maturation processes tend to be studied in a rather simplistic isolated manner, ample evidence exists demonstrating that the various events that constitute pre-mRNA maturation occur in an integrated fashion. Distinct machines carry out each of the steps in the gene expression pathway but despite the unique reactions they catalyse, each machine also interfaces both physically and functionally with other machines in the pathway. More specifically, a wide number of studies have highlighted the role of the Carboxyl Terminal Domain of the largest subunit of RNA Polymerase II as the key determinant player in the coupling of the pre-mRNA maturation events. In this work we have addressed the question of the co-ordination of all the steps of pre-mRNA processing using the poly(A) binding protein nuclear 1 (PABPN1) as a tool. PABPN1 is an abundant nuclear protein that stimulates poly(A) polymerase (PAP) during polyadenylation of mRNA precursors in metazoan cells. PABPN1 acts primarily as a stimulatory factor for the extension of the poly(A) tail and remains associated to the mRNA after the poly(A) addition, travelling with the mRNA to the cytoplasm where translation occurs. In addition to the very well characterized role in stimulating PAP, there are some other reports of additional PABPN1 functions. For instance, PABPN1 has been found in the nuclear nonsense-mediated decay complex, which is thought to complete a "pioneer" round of translation checking the integrity of the mRNA. PABPN1 has also been shown to associate with RNA Polymerase II along the chromatin axis and has been proposed to participate in transcription by associating with the Ski-interacting pre-spliceosomes, thus likely to be recruited protein and with MyoD. It has also been identified as a component of the spliceosome, neither stably associated with U4/U6.U5 tri-snRNPs nor present in purified to the spliceosome at a later, more active, stage of its assembly. Our results demonstrate that PABPN1 plays an unforeseen role in the splicing reaction. Combining a series of in vitro pulldown and immunoprecipitation assays, PABPN1 is shown to interact directly with very well characterized splicing factors and to be recruited to intronic sequences upon gene transcription activation. PABPN1 is also shown to modulate splice site choices in splicing reporter mini-genes, therefore implicating PABPN1, directly or indirectly, in alternative splicing regulation. In addition to its novel role in splicing, we also tried to understand the role of PABPN1 in a human genetic disease called Oculopharyngeal Muscular Dystrophy (OPMD). In 1998, it was found that mutations on the PABPN1 gene were responsible for OPMD. The mutation consists of short (GCG) expansions that result in the expansion of a polyalanine stretch at the N-terminus of the protein. Following the initial methionine, the wild type PABPN1 protein contains a naturally occurring trinucleotide repeat (GCG)6 coding for the first 6 alanines in a 10 tandem alanine stretch. In the OPMD patients, the (GCG)6 repeat is expanded to (GCG)8-13, thus leading to an expansion of the polyalanine tract from 10 to 12-17 alanines in the N-terminus of PABPN1. Because intranuclear inclusions containing PABPN1 represent a pathological hallmark of muscle cells from OPMD patients, it has been suggested that the mutated PABPN1 induces or facilitates the formation of the inclusions. To investigate whether this was the case, we have generated green fluorescent protein (GFP) fusions with PABPN1 variants. These fusion proteins were transiently expressed in human HeLa cells and in the murine myogenic cell line C2. We showed that, upon over-expression, PABPN1 aggregation can occur in the absence of polyalanine expansion. We further demonstrated that propensity of PABPN1 to form intranuclear inclusions is coupled to stimulation of polyadenylation, and protein aggregation is a highly dynamic, reversible process. Our results contributed greatly for the interpretation of OPMD model systems based on exogenous expression of PABPN1. |
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