Development of a RUBY-based visual pre-mRNA splicing reporter

Abstract

Precursor mRNA (pre-mRNA) splicing is a fundamental gene regulatory process in eukaryotes, with alternative splicing of multi-exon genes generating diverse transcript isoforms that control development and stress responses. Current splicing reporters based on fluorescent proteins, such as green fluorescent protein (GFP), or light-based luciferase have enabled in vivo analysis of splicing but require specialized equipment and/or costly substrates for signal detection. The RUBY reporter, which produces a visible red betalain pigment, was recently developed as a simple visual marker for a variety of purposes. Here, we repurposed RUBY as a splicing reporter, enabling visual detection of splicing without the need for expensive equipment or substrates. Three constructs were developed, each containing the genes CYP76AD1, DODA, and a glucosyltransferase (GT), which encode the three enzymes (cytochrome P450 monooxygenase, DOPA 4,5-dioxygenase, glucosyltransferase) required for betalain biosynthesis. The first construct contained only the enzymes required for betalain biosynthesis (RUBY), the second construct contained an intron within the coding region of the glucosyltransferase (RUBY-Intron), and the third construct contained the same intron with mutated splice sites in the coding region of the glucosyltransferase (RUBY-mIntronm). The constructs were first validated by transient expression in Nicotiana benthamiana leaves and subsequently used to generate at least two homozygous transgenic lines for each construct. Phenotypic analyses show that splicing of the intron in the RUBY-Intron construct enables betalain biosynthesis, resulting in a phenotype resembling that of RUBY. The mutated intron in RUBY-mIntronm results in a green phenotype like the wild type, as the mutated intron in the glucosyltransferase is not spliced, resulting in no expression of the glucosyltransferase and thus no betalain biosynthesis. The presence of each construct and the splicing of its transcript in transgenic lines were confirmed using genomic PCR and RT-PCR, respectively. Betalain content was quantified; RUBY and RUBY-Intron produced betalain, while RUBY-mIntronm did not, as expected. Splicing analyses revealed activation of cryptic splice sites in RUBY- mIntronm lines. The visual splicing reporter provides a cost-effective, noninvasive approach for studying splicing and opens new opportunities for forward genetic screens to identify splicing regulators and chemical inhibitors of splicing, with broad potential in both basic and applied research. Moreover, these constructs and transgenic lines could serve as valuable pedagogical tools in undergraduate laboratory courses for teaching the elaborations of the central dogma of molecular biology and highlighting the importance of RNA splicing. Betalain-based reporters, such as RUBY, have been widely used in recent years for diverse applications, including as transgenic markers, as reporters of hormone levels in planta, and for detecting environmental pollutants. However, the effects of RUBY expression and betalain production on plant development have not yet been systematically investigated. Here, I investigated the impact of RUBY expression at different stages of plant development. Arabidopsis plants constitutively expressing RUBY showed profound alterations in growth and reproduction. RUBY-expressing plants were significantly smaller, produced fewer leaves, exhibited delayed flowering, and often developed malformed siliques with reduced seed yield. In addition, root architecture was impaired, with markedly fewer lateral roots; this phenotype was only marginally rescued by indole-3-acetic acid. To discern the cause of developmental defects, we performed targeted metabolomic profiling, which revealed widespread alterations in amino acid and hormone levels, including reduced tryptophan, tyrosine, indole-3-acetic acid, and gibberellin, alongside accumulation of auxin precursors. These findings demonstrate that constitutive RUBY expression perturbs development and that some of the developmental defects are likely due to altered hormone and amino acid levels. These results highlight the importance of considering reporter-associated trade-offs and suggest a need for regulated inducible or tissue-specific expression for wider application of RUBY in plant research and biotechnology.