Challenges in Plant Alternative Splicing

Abstract

This chapter highlights the unique and common features of plant splicing compared to the better understood mammalian and yeast systems (see Chapter 5 L€ uhrmann and 6 Rymond for further discussion). In the past, experiments aimed at elucidating splicing mechanisms have been conducted mainly in mammalian and yeast systems, as these are more amenable to in vitro and genetic analyses. In fact, plant intron splicing began to attract more attention only when experiments conducted in planta indicated that plants were unable to splice out animal introns, or to recognize animal poly A signals correctly [1]. On the other hand, plant-derived introns were in most cases accurately and efficiently spliced in HeLa cells, using in vitro splicing extracts [2,3]. This prompted many lines of research to identify the mechanistic differences between splicing in plants, and splicing in animals. Consequently, although several laboratory groups embarked on the enterprise to develop an in vitro cell-based splicing extract, they invariably failed despite valiant attempts. Whilst plant cell extracts have been instrumental in elucidating the principles of translation, by using in vitro wheat germ system, it has not been possible to acquire a stable mRNA in vitro transcription or splicing system from plants. Hence, the detailed analysis of plant splicing has relied instead on the development of in vivo splicing analysis systems, that use transient transfection assays of splicing constructs and splicing factors (as discussed in Chapter 42, Simpson). The availability of the whole genome sequence of Arabidopsis has indicated that most of the important splicing components are also present in the plant genome [4]. Together with results from the in vivo analysis of splicing events in different plants, which indicated the requirement for U-rich sequences in plant introns, these data have indicated that the basic splicing mechanisms are comparable. However, there are clear differences in the size of plant introns compared to those of vertebrates, and also in the sequence composition of plant introns (U-rich). This, in turn, implies that there are differences in the definition of introns or exons, and thereby also differences in the RNA-binding properties of splicing regulatory proteins. Consequently, differences in intron composition and binding specificity, and the strength of splicing factors between different plant species as well as between plants and animals, might account for the variability in splicing efficiency.The past five years have witnessed a growing interest in alternative splicing in plants such that, today, high-throughput genomic and transcriptomic sequencing data are beginning to address the hitherto underdeveloped sequencing effort for expressed plant sequences [5] (see Chapter 23, Brown). Similar to mammalian systems (Chapter 3 Hertel), it has become very clear that alternative splicing is much more prevalent than was originally thought, rising from 7% to more than 35% in only three years as additional sequence data have become...

Rapid and Dynamic Alternative Splicing Impacts the Arabidopsis Cold Response Transcriptome

“…These include GEMIN2 (snRNP assembly), which is cold-induced, is involved in regulation of the circadian clock, and enhances U1snRNP assembly to compensate for reduced functionality of U1snRNP at low temperatures (Schlaen et al, 2015). Interestingly, a number of U1snRNP core and associated protein genes (U1-70k, LUC7B, LUC7RL, PRP39A, and RBM25) (Barta et al, 2012;Amorim et al, 2017;Kanno et al, 2017) respond rapidly to cold via AS. The early AS genes also include two wound-induced RNA binding proteins, UBA2a and UBA2c (Bove et al, 2008), and may also therefore be involved (A) Structures of highly expressed U2B"-LIKE transcripts (black boxes, UTR; color-coded boxes, exon coding sequence) and gene/transcript expression profile across the time course.…”

“…To identify putative splicing regulators/RNA binding proteins among the DE, DE+DAS, DAS, and TSIS genes, a reference list of Arabidopsis genes encoding RBPs, spliceosomal proteins, and SFs was assembled. First, published RRM-and KH-domain-containing RBPs from Lorković and Barta (2002) were combined with orthologs of human and yeast splicing proteins (Wang and Brendel, 2004;Barta et al, 2012) to give a nonredundant list of 317 SF-RBP genes. Recently, a protocol where proteins were cross-linked to RNA and then poly(A) + RNA was isolated and bound proteins were identified by proteomics/mass spectrometry was applied to Arabidopsis (Marondedze et al, 2016;Reichel et al, 2016).…”

Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

“…However, many aspects of pre-mRNA splicing in plants are yet to be elucidated. Furthermore, the composition of the plant spliceosome and its assembly intermediates are currently undefined [ 39 , 40 ]. Thus, the study of pre-mRNA splicing in plants requires innovative approaches, which will greatly empower this field of research.…”

Complexity of the Alternative Splicing Landscape in Plants

“…The analyses of splicing homologs in Arabidopsis have led to the identification of almost twice the number of splicing regulatory factors in comparison to human (see Supplemental Table 1 online) (Wang and Brendel, 2004;Barta et al, 2012;Koncz et al, 2012). The presence of multiple paralogs in plants seems to be the result of whole-genome duplications followed by massive genomic rearrangements in the course of evolution (Kalyna and Barta, 2004;Adams and Wendel, 2005;Cui et al, 2006).…”

“…The lack of an in vitro splicing assay in plants has been a major limitation in studying the mechanisms involved in intron recognition and spliceosome assembly in plants . However, the advent of the genomic era and the availability of whole-genome sequences of several plants allowed the identification of orthologous proteins and small nuclear RNAs (snRNAs) of the core components of the spliceosome (Wang and Brendel, 2004;Barta et al, 2012;Koncz et al, 2012) (see Supplemental Table 1 online), suggesting that the main principles of intron processing are also applicable to plants. Nevertheless, the fact that animal introns cannot be processed in plants made it clear that there is some specificity in the plant spliceosomal machinery and in the plant intronic sequences for their successful splicing Brown et al, 1986;Hartmuth and Barta, 1986).…”