A Dual Reporter Splicing Assay Using HaloTag-containing Proteins

Abstract

To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.

Keywords: CYBB gene; Chronic granulomatous disease; HaloTag fusion protein; genetic mutation.; luciferase reporter assay; splicing.

Fig. (1). Experimental overview

The inserted DNA fragments were designed to contain introns with or without mutations and the adjacent exons. The primers used to amplify the fragments in PCRs included 20-bp sequences that overlapped with the terminal sequences of the digested plasmid DNA. An In-Fusion reaction was used to ligate each DNA fragment to an exon/intron cassette-trapping plasmid containing the Renilla luciferase gene and HaloTag gene (pFN21A-RL). After the construct was transfected into mammalian cells, successful splicing produced HaloTag-luciferase fusion proteins and high levels of luciferase activity, whereas aberrant splicing or no splicing did not.

Fig. (2). Effects of mutations at splice sites in the BTK gene on splicing events

Splicing events in BTK transcripts were examined using RT-PCR products, luciferase activity, and HaloTag fusion proteins. We examined the effects of a G>T change at position –1 of the 3’-splicing acceptor site in intron 16 of the BTK gene. The experiment was carried out in triplicate and RT-PCR products were quantified as weighted ratios obtained from a MCE-202 MultiNA Microchip Electrophoresis System. Schematic representations of the RT-PCR products are shown on the right in the figure.

Fig. (3). Effects of mutations at splice sites in the WAS gene on splicing events

Splicing events in WAS transcripts were assessed using RT-PCR products, luciferase activity, and HaloTag fusion proteins. We used exon/intron cassette-trapping plasmids with or without mutations (G>A, G>T, G>C, or wild-type) at position +5 of the 5’-splicing donor site in intron 6 of the WAS gene. The experiment was carried out in triplicate and RT-PCR products were quantified as weighted ratios obtained from a MCE-202 MultiNA Microchip Electrophoresis System. Schematic representations of the RT-PCR products are shown on the right in the figure.

Fig. (4)Patient phenotypes and direct sequencing of the CYBB gene

A. Flow cytometry with DHR-123 demonstrated that reactive oxygen species were produced at low levels in neutrophils obtained from the patient. The effects of a null mutation (del425_426CT, 142S>fsX) are shown as a negative control. B. Flow cytometry with anti-cytochrome b₅₅₈ monoclonal antibodies showed markedly decreased surface expression of cytochrome b₅₅₈ on the patient’s neutrophils. C. Direct sequencing of the CYBB gene indicated that the patient had a homozygous single G>C nucleotide substitution at position +5 in the consensus 5’-donor splice site of intron 5.

Fig. (5). RNA analysis of neutrophils from a patient with CGD, his mother, and a control subject

Single-stranded cDNA was synthesized from neutrophil RNA obtained from the patient, his mother, and a control subject. cDNA was PCR-amplified using a sense primer specific for exon 4 and an antisense primer specific for exon 8 of the CYBB gene. The products were separated on a 3% agarose gel and stained with ethidium bromide. DNA size markers are shown on the left and the numbers on the right indicate differ-ently spliced products. Although some normally splice transcripts were identified, most PCR products amplified from the patient lacked exon 5 or exons 5 and 6. Normally spliced transcripts and transcripts lacking exon 5 were identified in the mother.

Fig. (6). Effects of splice-site mutations in CYBB exon/intron cassettes containing a single intron

Splicing events identified based on RT-PCR products, luciferase activity, and HaloTag fusion proteins are shown. We used exon/intron cassette-trapping plasmids with or without mutations (G>C, G>T, G>A, or wild-type) at position +5 of the 5’-splicing donor site in intron 5 of the CYBB gene. The experiment was carried out in triplicate and levels of RT-PCR products were quantified as the weighted ratios obtained using a MCE-202 MultiNA Microchip Electrophoresis System. Schematic representations of the RT-PCR products are shown on the right in the figure.

Fig. (7). Effects of splice-site mutations in CYBB exon/intron cassettes containing two introns

Splicing events identified based on RT-PCR products, luciferase activity, and HaloTag fusion proteins are shown. We used exon/intron cassette- trapping plasmids with or without mutations (G>C, G>T, G>A, or wild-type) at position +5 of the 5’-splicing donor site in intron 5 of the CYBB gene. The experiment was carried out in triplicate and levels of RT-PCR products were quantified as the weighted ratios obtained using a MCE-202 MultiNA Microchip Electrophoresis System. Schematic representations of the RT-PCR products are shown on the right in the figure.

Fig. (8). Effects of splice-site mutations in CYBB exon/intron cassettes containing three introns

Splicing events identified based on RT-PCR products, luciferase activity, and HaloTag fusion proteins are shown. We used exon/intron cassette-trapping plasmids with or without a mutation (G>C or wild-type) at position +5 of the 5’-splicing donor site in intron 5 of the CYBB gene. The experiment was carried out in triplicate and levels of RT-PCR products were quantified as the weighted ratios obtained from a MCE-202 MultiNA Microchip Electrophoresis System. Schematic representations of the RT-PCR products are shown on the right in the figure.