Publicação
Regulation of protein-coding gene expression by promoter-proximal convergent antisense transcription
| Resumo: | High-throughput sequencing technologies have shown that eukaryotic cells produce numerous noncoding transcripts. Among these are long non-coding RNAs (lncRNAs), defined as transcripts of more than 200 nucleotides that do not belong to any other well-defined group of non-coding RNAs. A particular class of lncRNAs overlaps protein-coding (PC) genes and are transcribed from the opposite strand, being known as natural antisense transcripts (NATs). A specific subgroup of NATs that overlap the promoter region of the PC genes with convergent transcription ending upstream of the PC gene transcription start sites were called promoter-proximal convergent antisense transcripts (PCATs). Studies on the transcriptional pair PC/PCAT, ZEB2/ZEB2- NAT, revealed that reducing ZEB2-NAT RNA levels results in the downregulation of ZEB2 transcripts. While this study highlights the physiological role of ZEB2-NAT in ZEB2 protein-encoding gene expression, the mechanism by which this occurs is not understood. The goal of this project was to test the hypothesis that ZEB2-NAT enhances the transcription of the overlapping PC gene ZEB2 and to dissect the mechanisms by which this PCAT regulates ZEB2 transcription, using human induced pluripotent stem cells as the study model. Previous results have shown that ZEB2-NAT transcripts are up regulated when iPSCs shift from self-renewal to lineage commitment before the level of ZEB2 increases. iPSCs were manipulated to up-regulate and downregulate ZEB2-NAT RNA levels to assess the outcome on transcription of the ZEB2 gene by RT-qPCR. The up regulation was performed with two approaches: transient transfection of a cloned ZEB2-NAT gene (cis-acting mechanism) and activation of the endogenous promoters using CRISPR-SAM (trans-acting mechanism). The downregulation of ZEB2-NAT was made with antisense LNA GapmeRs. The exogenous expression of ZEB2-NAT RNA either in iPSCs or during early mesoderm commitment did not increase ZEB2 mRNA levels. However, the activation of the endogenous ZEB2-NAT expression using CRISPR-SAM was able to induce the expression of ZEB2, but only in iPSCs and not during early mesoderm commitment. The antisense LNA GapmeRs against ZEB2-NAT RNA did not accomplish a significative reduction of its levels, precluding an effect on ZEB2 gene expression. Taken together the results obtained support the hypothesis that ZEB2-NAT function by enhancing the expression of the overlapping protein-coding gene ZEB2 and suggest this is mediated by a cis-acting mechanism. |
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| Autores principais: | Dias, Inês Gonçalves |
| Assunto: | Regulação da expressão genética lncRNAs ZEB2-NAT ZEB2 Teses de mestrado - 2023 |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | High-throughput sequencing technologies have shown that eukaryotic cells produce numerous noncoding transcripts. Among these are long non-coding RNAs (lncRNAs), defined as transcripts of more than 200 nucleotides that do not belong to any other well-defined group of non-coding RNAs. A particular class of lncRNAs overlaps protein-coding (PC) genes and are transcribed from the opposite strand, being known as natural antisense transcripts (NATs). A specific subgroup of NATs that overlap the promoter region of the PC genes with convergent transcription ending upstream of the PC gene transcription start sites were called promoter-proximal convergent antisense transcripts (PCATs). Studies on the transcriptional pair PC/PCAT, ZEB2/ZEB2- NAT, revealed that reducing ZEB2-NAT RNA levels results in the downregulation of ZEB2 transcripts. While this study highlights the physiological role of ZEB2-NAT in ZEB2 protein-encoding gene expression, the mechanism by which this occurs is not understood. The goal of this project was to test the hypothesis that ZEB2-NAT enhances the transcription of the overlapping PC gene ZEB2 and to dissect the mechanisms by which this PCAT regulates ZEB2 transcription, using human induced pluripotent stem cells as the study model. Previous results have shown that ZEB2-NAT transcripts are up regulated when iPSCs shift from self-renewal to lineage commitment before the level of ZEB2 increases. iPSCs were manipulated to up-regulate and downregulate ZEB2-NAT RNA levels to assess the outcome on transcription of the ZEB2 gene by RT-qPCR. The up regulation was performed with two approaches: transient transfection of a cloned ZEB2-NAT gene (cis-acting mechanism) and activation of the endogenous promoters using CRISPR-SAM (trans-acting mechanism). The downregulation of ZEB2-NAT was made with antisense LNA GapmeRs. The exogenous expression of ZEB2-NAT RNA either in iPSCs or during early mesoderm commitment did not increase ZEB2 mRNA levels. However, the activation of the endogenous ZEB2-NAT expression using CRISPR-SAM was able to induce the expression of ZEB2, but only in iPSCs and not during early mesoderm commitment. The antisense LNA GapmeRs against ZEB2-NAT RNA did not accomplish a significative reduction of its levels, precluding an effect on ZEB2 gene expression. Taken together the results obtained support the hypothesis that ZEB2-NAT function by enhancing the expression of the overlapping protein-coding gene ZEB2 and suggest this is mediated by a cis-acting mechanism. |
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