On February 9, 2022, Genome Biology published a research paper by the Shu-Hsing Wu team at the Academia Sinica in Taiwan entitled "An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development." The study identified more than 30,000 variable splicing gene models across 16,500 gene loci through PacBio isomer sequencing (Iso-seq) and revealed that the BBX splicing variant produced by intron retention regulates the developmental process of Arabidopsis thaliana photomorphogenesis.
https://doi.org/10.1186/s13059-022-02620-2
Light opens up the development of light morphology in plant seedlings, allowing seedlings to acquire the ability to photosynthesis and gain survival adaptability. The B-BOX domain protein BFX (B-BOX DOMAIN PROTEIN) can be expressed by light and plays an important role in photomorphogenesis. In addition to transcriptional activation and inhibition, variable splicing (AS) of mRNA is also one of the main ways to regulate gene expression. However, traditional short-read RNA sequencing techniques greatly limit the accurate determination of full-length (FL) splicing variants, and it remains unclear whether variants produced by variable splicing can regulate the developmental process of photomorphogenesis.
To answer this question, the study performed PacBio long-read Iso-seq sequencing on 4-day-old yellowing seedlings treated with 4 h dark or white light to fully identify full-length splicing variants. The researchers found more than 30,000 splicing variants from about 16,500 gene loci; of these, about 12,000 were new gene models. Further analysis found that most of the splicing variants of Iso-seq were consistent with transcriptome expression of the original gene (Figure 1). In addition, by comparing it with the TAIR10 database, Iso-seq sequencing also found that nearly 700 genes were new genes that had not been annotated.
Figure 1. Comparison of gene and splicing variant expression
The main types of variable splicing are intron retention, variable donor (altD) and variable acceptor (altA) sites, exon jump ES (exon skipping), covert intron CI (cryptic intron) and covert exon CE (cryptic exon). The study found that most splicing variants are produced by IR mode, about 34 percent, followed by variable donor or recipient sites, which account for about 30 percent.
So, do the variants produced by the variable splicing regulate the development of light morphology? The study found that all 212 transcription factorSTFs involved in photophylomorphism have variable splicing variants. Considering that transcription factors generally perform regulatory functions in the form of homologous or heterodimers, the researchers speculate that IR variants of TF may form heterodimers with TF. Using yeast double heterozygous, the study demonstrated that BBX22IR, BBX24IR, PIF3IR and PIF4IR variants can bind to their corresponding TFs (Figure 2).
Figure 2. The IR variant of TF can form heterodimers with TF
BBX22 is a positive regulator of photomorphogenesis; the study found that overexpressed plants of the BBX22IR variant exhibit photosensitivity phenotypes, and that BBX22IR may regulate gene expression in the form of homologous dimers in BBX22 mutants. In addition, BBX22 degrades in the dark or in prolonged light; unlike this, the BBX22IR has good stability (Figure 3 left).
Finally, the study found that overexpressed plants with BBX24IR exhibit short hypocotyls; suggesting that, contrary to the negative regulatory role of BBX24 in light morphogenesis, BBX24IR plays a positive regulatory role in this process (Figure 3 right).
Figure 3. Pattern diagram of the BBX variant that regulates light morphology
In summary, the study identified the presence of variable splicing at a large number of gene loci during photomorphogenesis through Iso-sequencing by PacBio, and revealed that IR variants of BBX22 and BBX24 regulate the developmental process of Arabidopsis photomorphogenesis. The methods used in this study provide new ideas and research frameworks for future studies of the function of variable splicing variants during development, response to environmental changes, and functional diversity in related species.
Original link:
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02620-2
Contributed by: SCI Shi
Editor-in-charge: Li Chen
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