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.

HaloTag Technology: A Versatile Platform for Biomedical Applications

“… 88 Similar techniques were utilized in the development of dual reporter genes for evaluating the process of mRNA splicing using the HaloTag platform. 89 …”

“…In conjunction with flow and laser scanning cytometry, statistical measurements of protein expression in individual or groups of cells can be analyzed . Similar techniques were utilized in the development of dual reporter genes for evaluating the process of mRNA splicing using the HaloTag platform …”

Rational Design of an Activatable Reporter for Quantitative Imaging of RNA Aberrant Splicing In Vivo

“…14 Several studies have designed fluorescent proteins or luciferase as splicing reporters to evaluate the mRNA that constitute splicing or alternative splicing events. [15][16][17][18][19] Although these reporter systems were able to successfully detect that the pre-mRNA splicing process responded to extracellular splicing modulators, some of the reporters were negative systems in nature, and people could not distinguish whether the reduced signal is attributed to cell death or endogenous splicing induced by the splicing compound. In addition, most of the splicing reporters exhibited poor dynamic range or fail to distinguish changes in compound-affected splicing from changes in mRNA transcription or translation.…”

HaloTag® Platform: From Proteomics to Cellular Analysis and Animal Imaging

“…Fluorescence intensities of protein bands following SDS-PAGE likely reflect the relative levels of HaloTag proteins after cell lysis. Oshima et al [ 17 ] combined the advantages of HaloTag and the Dual Luciferase Assay to quantitatively examine splice variants (i.e., unspliced, aberrantly spliced, and correctly) of mRNA products to determine relative splicing efficiencies with and without mutations represented in 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.…”