Elsevier

Gene

Volume 660, 20 June 2018, Pages 92-101
Gene

Research paper
The matrices and constraints of GT/AG splice sites of more than 1000 species/lineages

https://doi.org/10.1016/j.gene.2018.03.031Get rights and content

Highlights

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    A resource for matrices/constraints of GT/AG splice sites of Ensembl-annotated genomes

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    The matrices/constraints are highly diverse in a species-, genus- or phylum-specific way.

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    A bell-curve for the abundance of alternative splicing and the species constraint indexes of splice sites

Abstract

To provide a resource for the splice sites (SS) of different species, we calculated the matrices of nucleotide compositions of about 38 million splice sites from >1000 species/lineages. The matrices are enriched of aGGTAAGT (5′SS) or (Y)6N(C/t)AG(g/a)t (3′SS) overall; however, they are quite diverse among hundreds of species. The diverse matrices remain prominent even under sequence selection pressures, suggesting the existence of diverse constraints as well as U snRNAs and other spliceosomal factors and/or their interactions with the splice sites. Using an algorithm to measure and compare the splice site constraints across all species, we demonstrate their distinct differences quantitatively. As an example of the resource's application to answering specific questions, we confirm that high constraints of particular positions are significantly associated with transcriptome-wide, increased occurrences of alternative splicing when uncommon nucleotides are present. More interestingly, the abundance of alternative splicing in 16 species correlates with the average constraint index of splice sites in a bell curve. This resource will allow users to assess specific sequences/splice sites against the consensus of every Ensembl-annotated species, and to explore the evolutionary changes or relationship to alternative splicing and transcriptome diversity. Web-search or update features are also included.

Introduction

Splice sites (SS) demarcate exons and introns allowing the proper joining of exons during the expression of most eukaryotic genes. Their selective usage during alternative splicing produces more than one transcript from a single gene thereby contributing to transcriptomic and proteomic diversity (Black, 2003; Nilsen and Graveley, 2010). Their importance has been clearly demonstrated by splice site mutations that cause diseases (Tazi et al., 2009; Scotti and Swanson, 2016; Daguenet et al., 2015; Feng and Xie, 2013). Genomic analyses of different individual species/groups have given a glimpse of the consensus and diversity of both constitutive and alternative splice sites (Dou et al., 2006; Thanaraj and Stamm, 2003; Rogozin and Milanesi, 1997; Sibley et al., 2016; Szczesniak et al., 2013; Abril et al., 2005; Garg and Green, 2007; Burset et al., 2001). However, a centralized source for an overview of the annotated, millions of splice sites among all the currently sequenced eukaryotes remains to be created.

In biological or biomedical research, one often needs to assess the strength of particular sequences as splice sites or compare them between species, in fields such as genetics, cell biology, biochemistry or physiology. A resource with quantitative, comparable measurements of the splice site consensus and constraints of different species would be a very helpful reference. We thus compiled this resource for the consensus and diversity of the splice sites of the GT/AG introns of the Ensembl-annotated eukaryotic species as a reference for simple search or further exploration.

The GT/AG splice sites present in the majority of eukaryotic introns, characterized in humans with a consensus AGGTRAGT at the 5′ splice site and A(Y)nNYAGG (underlined: intron start/end GT/AG, A: branch point, Yn: polypyrimidine tract Py, N: A,C,G or T, n:6–35) at the 3′ splice site, with variations (except the GT/AG) in other species (Sibley et al., 2016; Moore, 2000; Burge et al., 1998; Spingola et al., 1999; Mount et al., 1992; Lorkovic et al., 2000). These sequences are recognized through direct base-pairing by the snRNAs of snRNP splicing factors (U1, U2, U5 and U6, with the participation of U4) or through contact by accessory proteins such as U2AFs during the dynamic assembly of spliceosomes (Will and Luhrmann, 2011; Shi, 2017). In this report, we used the Ensembl-annotated databases to compile a complete list of the matrices and constraints of the splice sites. Since the branch point is not as easy to assess accurately as the other motifs, it is not included here. Also not included are the minor AT/AC introns (<0.5%) (Burge et al., 1998; Verma et al., 2017; Wu and Krainer, 1999; Hall and Padgett, 1994; Levine and Durbin, 2001).

Section snippets

The matrices of GT/AG splice sites of >1000 eukaryotic species/lineages

We calculated the percent nucleotide compositions of the 5′ and 3′ GT/AG splice sites of 1074(5′)/1076(3′) species or their lineages/strains (hereafter ‘lineage’, to represent all in the same species, S_Tables I–II). An example of the resulting matrix format is shown in Fig. 1A, with the average percentages of >300 thousands of human splice sites. The matrices are enriched of (c/a)AGGT(A/g)AGt (5′SS) or (Y)20NCAGgt (3′SS, upstream beyond the (Y)20 is T/A-rich), similar to those based on about

A reference database for the splice site matrices/constraints of >1000 species/lineages

The matrix and constraint database of the annotated eukaryotic species has covered the 5′GT, 3′ polypyrimidine tract and 3′AG. It will be useful at least in the studies on genes or gene functions: as a reference for making effective mutations close to or within the splice sites in splicing assays, for assessing the strength of a particular splice site or mutant sequence in a species, for developing species-specific algorithm for more accurate prediction of unknown exons, for comparing splice

Genome data

The GenBank-format files of the genomes of all the species examined here were downloaded from the release 88 or Genome release 35 of the Ensembl database, of which the transcripts are based on experimental evidence (Aken et al., 2016).

Calculation of matrices and constraints

For matrices, the 5′ GT(−5–+50) or 3′ AG(−50–+2) splice sites were counted for their nucleotide compositions (percentages) at each position according to the intron coordinates in the annotated GenBank files of each species/lineage/strain, using Python scripts.

For

Acknowledgements

This work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2016-06004) and a Manitoba Research Chair fund to J.X. We thank the anonymous reviewers for helpful suggestions.

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