Publicação
Characterization of post-trancriptional control mechanisms regulating SMN2 gene expression
| Resumo: | Research has demonstrated that a significant part of gene expression control is taking place at the post-transcriptional level, determining the stability and translation efficiency of mRNA molecules. These regulatory switches are under the control of RNA-binding proteins and microRNAs that associate predominantly with the 5’ and 3’ UTRs. The prospect for development of effective therapies for Spinal Muscular Atrophy is enhanced by the existence of an almost identical copy of the SMN1 gene-mutated in SMA. The SMN2 gene can produce the functional protein at low levels; however variations in its copy number and thus, protein expression output have been shown to modulate disease severity in affected individuals. Although it is clear that the control of mRNA translation and stability can influence dramatically the protein output of a given gene, and in spite of the advantages that increased SMN2 expression may provide to patients, no systematic study has thus far been performed on the post-transcriptional control mechanisms acting on the SMN2 mRNA. Therefore, the identification of these mechanisms is of great interest, as it may provide the basis for new therapies for SMA. To identify and characterize the SMN2 post-transcriptional mechanisms regulating the SMN2 mRNA we established a Luciferase reporter system for monitoring UTR-dependent regulatory events. We verified that the SMN2 3’UTR region has a regulatory role in the mRNA stability, which results from a balance of positive and negative cis-acting elements. We also identified key conserved nucleotides in the 3’UTR, whose mutation resulted in increased mRNA expression levels. Analysis of the SMN2 5’UTR region indicates that this region has a negative regulatory effect on the SMN2 expression due to presence of a uORF. We additionally performed systematic validation of novel predicted alternative SMN2 5’ and 3’UTR sequences. We were able to validate the expression of an alternative 5’UTR isoform and obtained preliminary evidence for the regulation of SMN transcripts by the Nonsense Mediated Decay pathway. |
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| Autores principais: | Gomes, Ana Luísa Rodrigues, 1987- |
| Assunto: | Biologia molecular Expressão génica Atrofia muscular Teses de mestrado - 2010 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Research has demonstrated that a significant part of gene expression control is taking place at the post-transcriptional level, determining the stability and translation efficiency of mRNA molecules. These regulatory switches are under the control of RNA-binding proteins and microRNAs that associate predominantly with the 5’ and 3’ UTRs. The prospect for development of effective therapies for Spinal Muscular Atrophy is enhanced by the existence of an almost identical copy of the SMN1 gene-mutated in SMA. The SMN2 gene can produce the functional protein at low levels; however variations in its copy number and thus, protein expression output have been shown to modulate disease severity in affected individuals. Although it is clear that the control of mRNA translation and stability can influence dramatically the protein output of a given gene, and in spite of the advantages that increased SMN2 expression may provide to patients, no systematic study has thus far been performed on the post-transcriptional control mechanisms acting on the SMN2 mRNA. Therefore, the identification of these mechanisms is of great interest, as it may provide the basis for new therapies for SMA. To identify and characterize the SMN2 post-transcriptional mechanisms regulating the SMN2 mRNA we established a Luciferase reporter system for monitoring UTR-dependent regulatory events. We verified that the SMN2 3’UTR region has a regulatory role in the mRNA stability, which results from a balance of positive and negative cis-acting elements. We also identified key conserved nucleotides in the 3’UTR, whose mutation resulted in increased mRNA expression levels. Analysis of the SMN2 5’UTR region indicates that this region has a negative regulatory effect on the SMN2 expression due to presence of a uORF. We additionally performed systematic validation of novel predicted alternative SMN2 5’ and 3’UTR sequences. We were able to validate the expression of an alternative 5’UTR isoform and obtained preliminary evidence for the regulation of SMN transcripts by the Nonsense Mediated Decay pathway. |
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