Importance of pre-mRNA splicing and its study tools in plants

Abstract

Alternative splicing (AS) significantly enriches the diversity of transcriptomes and proteomes, playing a pivotal role in the physiology and development of eukaryotic organisms. With the continuous advancement of high-throughput sequencing technologies, an increasing number of novel transcript isoforms, along with factors related to splicing and their associated functions, are being unveiled. In this review, we succinctly summarize and compare the different splicing mechanisms across prokaryotes and eukaryotes. Furthermore, we provide an extensive overview of the recent progress in various studies on AS covering different developmental stages in diverse plant species and in response to various abiotic stresses. Additionally, we discuss modern techniques for studying the functions and quantification of AS transcripts, as well as their protein products. By integrating genetic studies, quantitative methods, and high-throughput omics techniques, we can discover novel transcript isoforms and functional splicing factors, thereby enhancing our understanding of the roles of various splicing modes in different plant species.

Keywords: Alternative splicing; Modern biotechnology; Plant development; Splicing machinery; Stress response.

Fig.1

A possible AS regulation network exists for plant development, circadian rhythms, and stress response. Due to the Earth's rotation, all organisms are influenced by changes in light intensity. As a result, most species have developed an endogenous circadian clock that operates on a roughly 24-h cycle, known as the circadian rhythm. Splice-related factors, including spliceosome complexes, splicing factors, and functional proteins, target proper splicing of a serious of functional genes to regulate plant development, circadian and responses to abiotic stress. Different colored boxes represent exons and lines represent introns

Fig. 2

Representation of modern techniques for the study of alternative spliced transcripts and their protein products. A To understand the function of isoforms, obtaining the gene mutants with gain of function and knock-out is necessary. The genetic transformation of A. thaliana (representing dicotyledon) and maize (representing monocotyledon) are shown separately. Maize mutant lines require the co-culture of Agrobacterium tumefaciens carrying a target expression vector with immature corn embryos, followed by subsequent tissue culture. The corresponding A. thaliana mutant is created by infecting unfertilized inflorescence with Agrobacterium tumefaciens carrying target vector. P35S, CaMV 35S; Pubi, ubiquitin promoter. B QuantAS, an absolute quantitative method for splicing variants that combines qRT-PCR and dPCR. C Several high-throughput omics technologies. Spliced transcripts and their corresponding protein products can be obtained by combining second-generation transcriptome sequencing, represented by Illumina, with third-generation sequencing technologies, represented by Nanopore and PacBio, coupled with LC–MS/MS analysis. DEG, differentially expressed gene; DEP, differentially expressed protein; DAS, differentially expressed AS; DASDP, differentially expressed AS peptide