Design of a GFP reporter for splicing analysis in mammalian cells

Abstract

Eukaryotic genes are formed by exons and introns. Pre-mRNA splicing promotes exon ligation and intron removal and is performed by a specialized macromolecular machinery named spliceosome, composed of five small ribonucleoprotein particles (snRNPs) and more than one hundred proteins. The activity of this complex is highly accurate due to the coordinated activity of its components. Altered splicing has been related to the development of several diseases, including neurodegenerative disorders, such as amyotrophic lateral sclerosis, and different types of cancer. Detailed understanding of splicing regulation in eukaryotic cells can be achieved using splicing reporter systems. We designed a reporter plasmid suitable for splicing analysis in cultured mammalian cells. Our reporter is based on GFP expression, and the splicing outcome can be easily visualized by fluorescence microscopy. We quantified splicing activity in two human cell lines, HEK-293T and MDA-MB-231, confirming its suitability for use in live cells in culture.

Keywords: GFP; Pre-mRNA splicing; Reporter system.

Fig. 1

pGFP-spl plasmid construction and fluorescence analysis of GFP expression in HEK-293T live cells. (A) The generation of pGFP-spl is shown, with the cassette containing GFP exons (green) and the intron (blue) (843 bp). Total length of spliced GFP exons (“mRNA”) is 720 bp, and intron length is 123 bp. (B) Fluorescence after 24 h and 48 h of transfection is shown on the left and bright field is on the right. Magnification 40x. Mean fluorescence intensity quantification from approximately 100 cells are shown on the right-hand side. pGFP-spl mean fluorescence intensity was quantified after 24 h and 48 h of transfection using Fiji software. Statistical analysis was performed by unpaired T-test (GraphPad Prism 8.0.2). ***p < 0.001. Further fields are shown in Supplementary Figure 2 and raw images are available at https://github.com/PColtri/Menezes2025/.

Fig. 2

PCR and RT-qPCR analysis of splicing. HEK-293T cells were transfected with pGFP-spl and pEGFP (Invitrogen) as a positive control. (A) Electrophoresis of PCR products. PCR reactions were performed using cDNA as template and primers to detect the transcripts corresponding to total RNA (odd lanes, PC155/PC156) and mRNA (even lanes, PC157/PC156). The schemes at the bottom show the primer pairs annealing to amplify total RNA, pre-mRNA and mRNA. Red arrowheads on the left point to reference size bands for comparison. MW: Molecular weight ladder 1 kb (Thermo). (B) Graph showing fold change in mRNA levels after normalization with β-actin and using pEGFP as standard. pGFP-spl and pEGFP (positive control) are shown. Standard deviations were calculated after two technical replicates. Statistical analysis was performed by unpaired T-test (GraphPad Prism 8.0.2). *p < 0.01. In the lower part of the figure, the raw Ct mean for each sample and with both sets of primers are shown.

Fig. 3

Time-course analysis of splicing reactions using the pGFP-spl reporter. Samples of (A, B) HEK-293T and (C, D) MDA-MB-231 cell lines were analyzed. Cells were collected 1, 3 and 24 h after transfection using pGFP-spl and analyzed by RT-qPCR using primers for (A, C) mRNA and (B, D) pre-mRNA. In comparison to levels observed 1 h after transfection, fold change was calculated (y-axis). Statistical analysis was performed by One-way ANOVA with Tukey's post-hoc (GraphPad Prism 8.0.2). Only statistically significant differences are indicated. *p < 0.01; **p < 0.001; ***p < 0.0001.