Abstract |
In recent years, a shift in research focus has been observed, with a transition from mRNAs to non-coding RNAs, highlighting their crucial roles in the regulation of gene expression. Among those RNA species, long non-coding RNAs (lncRNAs), characterized by lengths exceeding 200 nucleotides, have gained prominence. These lncRNAs are implicated in gene regulation through interactions with proteins and other RNAs. These interactions are often associated with specific sequence motifs and features, such as splicing. Despite their relatively low splicing efficiency, lncRNAs exhibit a high degree of alternative splicing, resulting in the generation of various isoforms that can profoundly impact cellular functions and contribute to disease development, including cancer. Notably, Plasmacytoma Variant Translocation 1 (PVT1), located on chromosome 8 in the human genome and positioned closely to the MYC gene, has emerged as a prominent lncRNA. Its expression has been linked to various cancer types, including breast cancer, a leading cause of cancer-related deaths in women. Recent studies have characterized PVT1 as a lncRNA that is retained within chromatin. Machine learning experiments have suggested an association between splicing efficiency and chromatin association. Therefore, the aim of this study is to enhance the splicing efficiency of PVT1, in the worst processed 3’ splice sites, as indicated by the bioinformatic analysis of RNA-seq data for MCF-7 cell lines (representing luminal A breast cancer) and MCF-10A/MCF-12A (control) cell lines. To achieve splicing enhancement, the CRISPR Artificial Splicing Factors (CASFx) tool, a modified version of CRISPR-Cas9 containing as splicing activator the fused protein RBFOX1-dCasRx, is employed. A modified CASFx vector has been successfully constructed, allowing for the insertion of guide RNAs for any target in the transcriptome. Initial experiments have revealed promising results, suggesting that the constructed plasmid can influence exon inclusion and potentially impact MYC gene expression levels. However, further investigations are necessary to elucidate the tool's effect on PVT1 chromatin association, and further evaluate its downstream effects. This study holds the potential to uncover critical insights into the role of PVT1 in breast cancer and may open avenues for novel therapeutic approaches.
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