Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts

Abstract

The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates.

FIGURE 1.

Structure of hnRNPs A/B at gene, transcript, and protein level. (A) Comparison of exons in A1*, A2/B1, and A3 in mouse mRNA and approximate alignment with protein domains. A1* is alternatively spliced to produce the transcripts A1 and A1b, A2/B1 is spliced to produce transcripts B1, A2, A2b, and B1b, and A3 is spliced to produce transcripts A3a and A3b. Each numbered rectangle represents an exon, with lengths adjusted to align homologous exons. Vertical dashed lines highlight the structural similarity in terms of exon lengths, the correspondence of exons to functional protein domains, and the location of alternative exons relative to the entire mRNA. Major transcripts (A1, A2, and A3b) are in boldface. Alternative exons are in a darker shade. RRM, RNA recognition motif; GRD, glycine-rich domain; M9, nuclear localization domain. (B) Splicing of alternative exons in hnRNPs A/B in mouse. Boxes represent exons and black horizontal lines represent introns. Bold blue and faint green lines represent major and minor splicing patterns, respectively. Note that exon 7b is mostly excluded from A1* exon 7b, while the corresponding exon 9 in A2/B1 is generally included and exon 8 in A3 appears to be constitutively present in mouse.

FIGURE 2.

Pairwise alignment of hnRNP A/B genes in different species. Red and green regions correspond to sections of high and low identity, respectively. The black bars under the heatmaps represent the locations of exons within the genes. The exon numbering is based on that used in Figure 1, and the exon size and numbering correspond to that of the species or paralog in boldface. The numbers for alternative exons are in blue. Additional heatmaps can be viewed in Supplemental Figure S2.

FIGURE 3.

Changes in splice site strength Splice site strengths of C-terminal AEs in different species. The 5′ss sequence flanking A1 exon 7b differs between Xenopus and the other species at the +4 position (arrow).

FIGURE 4.

Expression patterns of alternative exons in different species. (A) RT-PCR analysis of inclusion levels of alternative exons of hnRNPs A/B in brain cDNA from different species (labels as in Fig. 3). Arrows point to novel amplicons not previously described, while asterisks indicate presence of double bands due to band-splitting. Ratios of band intensities of amplicons that have variable proportions across mammals are summarized in Table 1. (B) Splicing of alternative exons in hnRNP A3 in human and chicken. Boxes represent exons and black horizontal lines represent introns. Bold blue and faint green lines represent major and minor splicing patterns, respectively. Novel splicing patterns identified in the RT-PCR analysis are dashed.

FIGURE 5.

Effect of alternative exons on protein function and expression. (A) Binding properties: Western blots of rat whole-brain lysate (WBL), and A2RE11- and Telo4-binding proteins (A and T, respectively) isolated from rat brain by affinity pulldowns (PDs). The ratio of band intensities within a lane reflect the ratio of isoform protein levels present in the sample loaded. The samples for A3a and A3b were run on the same gel separated by a lane for the protein size marker (M), which has been included in the images to ensure accurate alignment of the lanes and correct assignment of isoform identities. Anti-A3^NT recognizes A3a only, while anti-A3^CT recognizes both A3a and A3b. (B) A3b is not expressed in human: Whole-cell lysate from SH-SY5Y cells, a human neuroblastoma cell line immunostained with anti-A3^NT and anti-A3^CT. A3b was not detected under these loading and staining conditions. (C) Translation efficiency: FACS measurements of pEGFP levels in HeLa cells transfected with pEGFP expression vectors containing exon 1b or 2 of A3. §P < 0.001 by single factor ANOVA, *P < 0.05 by least significant difference method (n = 3). Bars represent standard deviation.