Identification and analysis of alternative splicing events in Phaseolus vulgaris and Glycine max

Luis P Iñiguez,William B Barbazuk,Georgina Hernández,Mario Ramírez

doi:10.1186/s12864-017-4054-2

Abstract

BackgroundThe vast diversification of proteins in eukaryotic cells has been related with multiple transcript isoforms from a single gene that result in alternative splicing (AS) of primary transcripts. Analysis of RNA sequencing data from expressed sequence tags and next generation RNA sequencing has been crucial for AS identification and genome-wide AS studies. For the identification of AS events from the related legume species Phaseolus vulgaris and Glycine max, 157 and 88 publicly available RNA-seq libraries, respectively, were analyzed.ResultsWe identified 85,570 AS events from P. vulgaris in 72% of expressed genes and 134,316 AS events in 70% of expressed genes from G. max. These were categorized in seven AS event types with intron retention being the most abundant followed by alternative acceptor and alternative donor, representing ~75% of all AS events in both plants. Conservation of AS events in homologous genes between the two species was analyzed where an overrepresentation of AS affecting 5’UTR regions was observed for certain types of AS events. The conservation of AS events was experimentally validated for 8 selected genes, through RT-PCR analysis. The different types of AS events also varied by relative position in the genes. The results were consistent in both species.ConclusionsThe identification and analysis of AS events are first steps to understand their biological relevance. The results presented here from two related legume species reveal high conservation, over ~15–20 MY of divergence, and may point to the biological relevance of AS.

Highlights

The vast diversification of proteins in eukaryotic cells has been related with multiple transcript isoforms from a single gene that result in alternative splicing (AS) of primary transcripts
Four different types of AS events are the most frequently described in the literature: exon skipping (ES), where a whole exon is missed in comparison to the primary transcript; intron retention (IR), an intron is not spliced and is part of the mature mRNA; alternative donor (AD), the donor site, known as 5′ splicing site, change in the mRNA isoform; and alternative acceptor (AA), where the 3′ splicing site is different
Based on a primary transcript three additional AS events can be described; alternative splicing sites (ASS), where both donor and acceptor sites change; new intron (NI), when a splicing site appears in a reported exon; and retained exon (RE), a new exon replaces a previously annotated intron in the mature mRNA

Summary

Introduction

The vast diversification of proteins in eukaryotic cells has been related with multiple transcript isoforms from a single gene that result in alternative splicing (AS) of primary transcripts. The majority of protein-coding genes from eukaryotic organisms contain introns, non-coding sequences that need to be spliced from the primary transcript to generate mature functional mRNAs. some introns can be self-spliced, most require a spliceosome, specialized splicing machinery. Spliceosomes are large ribonucleoprotein complexes that include small nuclear RNAs (snRNA) [1,2,3]. The U1 snRNA recognizes signals from the 5′ splice site, a GT dinucleotide. The U2 snRNA recognizes the 3′ splice site that includes an AG dinucleotide, an adenine which functions as a branching point upstream

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Conclusion