Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins

Abstract

Alternative splicing is an important mechanism for fine tuning of gene expression at the post-transcriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarly positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins.

Figure 1

Schematic representation of atRSp31, the alternatively spliced products and the deduced proteins The domain structure of the atRSp31 protein is shown; the RNA recognition motifs (RRM, containing the highly conserved RNP2 and RNP1 sequences) and the arginine/serine (RS) regions are indicated. Exons are illustrated as boxes and intron sequences are shown as lines. Exons containing the 5′- and 3′-UTRs are painted grey, the coding region black and alternatively spliced exons are striped. The thin lines connect the protein domains with the gene structure indicating where intron insertions occur. The star indicates the new stop codon in the alternatively spliced products. mRNA3 contains intronic sequences of the 3′ alternatively spliced second and third intron; mRNA2 contains sequences of the 3′ and 5′ alternatively spliced second intron and the 3′ alternatively spliced third intron; mRNA1 is the generic spliced mRNA. Protein structures for the different splice forms are shown to the right of the corresponding mRNA species. Parts of proteins generated by included intronic sequences are shown as stripped boxes. RT–PCR analysis of RNA isolated from Arabidopsis flowers is shown next to schemes of corresponding splice variants.

Figure 2

Regulation of alternative splicing in atRSp31. (A) Analysis of atRSp31 splicing in Arabidopsis cell culture protoplasts. Protoplasts were transiently transformed with either the cDNA or the genomic construct of atRSp31 under 35S CaMV promoter (lanes 1 and 2). Lanes 3 and 4: control transformations with empty vector and water, respectively. Total RNA was analysed by RT–PCR. Upper panel: detection of the transgenic atRSp31 using the primer to HA tag and the primer to the fourth exon of atRSp31. Middle panel: detection of the endogenous atRSp31 using the primer to the sequence which is disrupted in transgene by insertion of HA tag and the primer to the fourth exon of atRSp31. Lower panel: RT–PCR of ubiquitin used as a loading control. (B) Immunodetection of HA-tagged atRSp31. Arabidopsis cell culture protoplasts were transformed with HA-tagged the cDNA or the genomic construct of atRSp31 under 35S CaMV promoter (lanes 1 and 2, respectively). Total protein extracts were analysed by western blot using a monoclonal antibody against HA-tag. Lane M, molecular weight markers. (C) Regulation of alternative splicing in atRSp31 by atRSZ33. Upper panel: total RNA from wild type (lane 1) and seedlings overexpressing atRSZ33 (lane 2) was analysed by RT–PCR using the primers to the beginning of the second and the end of the fourth exons of atRSp31. Lower panel: RT–PCR of ubiquitin used as a loading control.

Figure 3

Conservation of alternative splicing events in the RS plant-specific subfamily. (A) Gene structures and alternative splicing. Domain structure typical for this subfamily of proteins is shown on the top. Gene structures are shown in green. ESTs/cDNAs representing each type of splicing are shown in red. Long introns are boxed. Blue and green arrows indicate conserved alternative 3′ and 5′ splice sites, respectively. Black arrow in atRSp31a indicates the presence of the conserved sequence of the alternative 3′ splice site; however, in this case alternative splicing is not supported by EST/cDNA data. (B) Alignments of the nucleotide sequences around 3′ alternative splice site in the long introns and around the 3′ splice site of a constitutively spliced exon. Sequence of atRSp31a at the alternative 3′ splice site is shown in italics to indicate the lack of EST data supporting this splicing event. (C) Alignments of nucleotide sequences around the 5′ alternative splice site in the long introns and around the 5′ splice site of a constitutively spliced exon. Intronic and exonic sequences are shown in small and capital letters, respectively. Nucleotides identical at all positions are shaded.

Figure 4

An alignment of protein sequences and intron positions in the RS plant-specific subfamily Identical or similar amino acids are shaded in black or grey, respectively. RNP2 and RNP1 submotifs of the first and second RRMs are overlined. Positions and phases of introns are indicated by a color code: phase 0, blue; phase 1, red; phase 2, green. P.taeda intron positions are indicated only when supporting evidence is present. Position of the conserved alternatively spliced intron between RNP2 and RNP1 is indicated by an arrow. Alignment of the C-termini of proteins covering the RS region is not shown. at, A.thaliana; os, O.sativa; pt, P.taeda; pp, P.patens; cr, C.reinhardtii.

Figure 5

Conservation of alternative splicing events in the RS2Z subfamily (A) Gene structures and alternative splicing. Domain structure typical for this subfamily of proteins is shown on the top. Gene structures are shown in green. ESTs/cDNAs representing each type of splicing are shown in red. Long introns are boxed. Blue arrows indicate the conserved alternative 3′ splice sites. The black arrow in ppRSZ38 indicates the presence of the conserved sequences around the alternative 3′ splice site; however, alternative splicing is not supported by EST data. (B) Alignments of nucleotide sequences around the 3′ alternative splice site in the long introns and around a 3′ constitutive splice site. Sequence of ppRSZ38 at the alternative 3′ splice site is shown in italics to indicate the lack of EST data supporting this splicing event. Intronic and exonic sequences are shown in small and capital letters, respectively. Nucleotides identical at all positions are shaded.

Figure 6

An alignment of protein sequences and intron positions in the RS2Z plant-specific subfamily Identical or similar amino acids are shaded in black or grey, respectively. RNP2 and RNP1 submotifs of the RRM are overlined. Cysteine and histidine residues of zinc knuckles are underlined. Alignment of the C-termini of the proteins is not shown. Positions of introns are indicated by colour code: phase 0, blue; phase 2, green. P.taeda intron positions are indicated only when supporting evidence is present. Position of the conserved alternatively spliced intron between RNP2 and RNP1 is indicated by an arrow. at, A.thaliana; os, O.sativa; pt, P.taeda; pp, P.patens.