Browsing by Author "Rosado, Telma"
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- RAC1B alternative splicing regulation in colorectal cancer cellsPublication . Rosado, Telma; Gonçalves, Vânia; Jordan, PeterColorectal cancer (CRC) is a common malignancy with significant mortality, which presents different subtypes due to genetic heterogeneity. The focus of this work was on a subtype that is characterized by microsatellite instability and mutations in the BRAF gene, being associated, in most cases, with RAC1B overexpression. RAC1B is a splicing variant of the RAC1 gene and is characterized by the inclusion of an additional exon through alternative splicing, designated as exon 3b, presenting some regulation and signalling differences when compared to RAC1. RAC1B has been described as a promoter of tumour progression, therefore, it is considered as a potential therapeutic target, as it may allow the design of targeted therapies for patients with this subtype of CRC, but for this, understanding in more detail the mechanisms of RAC1B regulation is extremely important. Previous research projects in the host lab had already identified SRSF1 and SRSF3 as two splicing factors involved in the regulation of RAC1B splicing in CRC, so the first goal of this thesis work was to determine the effect of other selected proteins on the expression of RAC1B in colorectal cell lines. The proteins of interest are a nucleoporin, RANBP2, and four splicing factors, PTBP1, ESRP1, DIS3L2 and hnRNP U, which were previously described as upregulated in CRC or involved in the RAC1B splicing process in other cell lines. The effect of these proteins on RAC1B alternative splicing was assessed through their overexpression and depletion in two colorectal cell lines, NCM460 and HT29, and the analysis of RAC1 exon 3b inclusion was made at the transcript level by RT-PCR, and at the protein level through SDS-PAGE and Western blot. The obtained results provided strong evidence that, in fact, these proteins influence RAC1B expression, both at the transcript and protein level. Although the overexpression experiments performed in this work did not lead to many clear conclusions about the effect of the proteins of interest on the RAC1B expression, the siRNA assays were rather conclusive. Taking into account the results obtained through depletion experiments, RANBP2 seems to act as a RAC1B inhibitor in NCM460 cells, while PTBP1, ESRP1, DIS3L2 and hnRNP U are probably enhancers of that RAC1 isoform. In the HT29 cell line, RANBP2 and DIS3L2 were pointed as inhibitors of exon 3b inclusion, while PTBP1, ESRP1 and hnRNP U are most likely enhancers of that alternative splicing event. The second goal of this work was to identify PTBP1 and ESRP1 RNA-binding motifs in the RAC1 transcript sequence. Here, two predicted ESRP1 binding motifs in the RAC1 transcript were identified and mutated in a RAC1 minigene, in order to verify if any of these changes could avoid ESRP1 binding and consequently affect the splicing process. The presence of the mutations was confirmed through DNA sequencing, however, it was not possible in the remaining time to reclone the mutant fragments into the original minigene vector, to assure they did not have undesired mutations, allowing to proceed to the planned experiments. Future studies should be performed in order to confirm the obtained results, as not all of them resulted in statistically significant variations on RAC1B expression levels, and it is also imperative to clarify how the nucleoporin RANBP2 affects RAC1B expression and what binding sites are recognized by the studied splicing factors in the RAC1 pre-mRNA.