Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis

Abstract

Background: Recently, genomic sequencing efforts were finished for Oryza sativa (cultivated rice) and Arabidopsis thaliana (Arabidopsis). Additionally, these two plant species have extensive cDNA and expressed sequence tag (EST) libraries. We employed the Program to Assemble Spliced Alignments (PASA) to identify and analyze alternatively spliced isoforms in both species.

Results: A comprehensive analysis of alternative splicing was performed in rice that started with >1.1 million publicly available spliced ESTs and over 30,000 full length cDNAs in conjunction with the newly enhanced PASA software. A parallel analysis was performed with Arabidopsis to compare and ascertain potential differences between monocots and dicots. Alternative splicing is a widespread phenomenon (observed in greater than 30% of the loci with transcript support) and we have described nine alternative splicing variations. While alternative splicing has the potential to create many RNA isoforms from a single locus, the majority of loci generate only two or three isoforms and transcript support indicates that these isoforms are generally not rare events. For the alternate donor (AD) and acceptor (AA) classes, the distance between the splice sites for the majority of events was found to be less than 50 basepairs (bp). In both species, the most frequent distance between AA is 3 bp, consistent with reports in mammalian systems. Conversely, the most frequent distance between AD is 4 bp in both plant species, as previously observed in mouse. Most alternative splicing variations are localized to the protein coding sequence and are predicted to significantly alter the coding sequence.

Conclusion: Alternative splicing is widespread in both rice and Arabidopsis and these species share many common features. Interestingly, alternative splicing may play a role beyond creating novel combinations of transcripts that expand the proteome. Many isoforms will presumably have negative consequences for protein structure and function, suggesting that their biological role involves post-transcriptional regulation of gene expression.

Figure 1

Diagrams of the nine classes of alternative splicing. These are: Alternate Acceptor (AA), Alternate Donor (AD), Alternate Terminal Exon (ATE), Retained Exon and Skipped Exon (RE/SE), Initiation within an Intron (IWI), Termination within an Intron (TWI), and Spliced Intron and Retained Intron (RI/SI). The black boxed regions represent the coding region while the white boxed regions represent the 5' and 3' UTR regions. Introns are represented by a single line.

Figure 2

Histograms displaying the transcript support for three of the alternatively spliced isoforms in rice. The legend for the histogram is alternate acceptor (black bar), alternate donor (stippled bar), and alternate terminal exon (diagonally hatched).

Figure 3

Histograms displaying the transcript support for three of the alternatively spliced isoforms in Arabidopsis. The legend for the histogram is alternate acceptor (black bar), alternate donor (stippled bar), and alternate terminal exon (diagonally hatched).

Figure 4

Histograms displaying the occurrence of the alternate acceptor class by difference in sequence length between the two alternatively spliced acceptor sites. The histogram shows the bins for the range from 2 bp to 20 bp for rice (black bar) and Arabidopsis (stippled bar).

Figure 5

Histograms displaying the occurrence of the alternate donor class by differences in sequence length between the two alternatively spliced donor sites. The histogram shows the bins for the range from 2 bp to 20 bp for rice (black bar) and Arabidopsis (stippled bar).

Figure 6

Pictogram illustrating the nucleotide sequence flanking the 3' splice site acceptor in rice. This analysis was done using the sequence from the alternate acceptor class with a sequence length difference between the two isoforms ranging from 2 bp to 10 bp. The consensus acceptor site is shown at the top (Con).

Figure 7

Pictogram illustrating the nucleotide sequence flanking the 5' splice site donor in rice. This analysis was done using the sequence from the alternate donor class with a sequence length difference between the two isoforms ranging from 2 bp to 10 bp. The consensus acceptor site is shown at the top (Con).