SR proteins and the nonsense-mediated decay mechanism are involved in human GLB1 gene alternative splicing

Abstract

Background: The human GLB1 gene is known to give rise to two alternatively spliced mRNAs, which encode two different proteins: lysosomal beta-galactosidase (beta-gal) and elastin-binding protein (EBP). The beta-gal transcript includes the 16 exons of the GLB1 gene. In the EBP transcript, exons 3, 4 and 6 are skipped, while exon 5 has a different reading frame. However, little is known on how this alternative splicing is regulated.

Findings: Cycloheximide treatment of HeLa cells and human fibroblasts revealed the presence of new transcripts that are otherwise degraded by nonsense-mediated decay (NMD). A minigene carrying the exons involved in the alternative splicing of GLB1 was constructed. Improving the acceptor-site scores of exons 3 or 4 increased the relative inclusion of these exons, but did not stop them being skipped in some transcripts. Overexpression of different SR proteins altered the relative proportion of the different transcripts produced by the minigene, indicating a possible mechanism for the regulation of the alternative splicing of GLB1. Finally, a comparison of this gene among different species was performed.

Conclusion: In the processing of the GLB1 RNA several transcripts are generated, but only those with a correct reading frame are not degraded. The differential inclusion/exclusion of exons could be partially explained by the relatively weak splice sites in the exons involved. Different SR proteins have an effect on the process of skipping of these exons, at least in the minigene conditions, indicating a possible mechanism for the regulation of the alternative splicing of the GLB1 gene.

Figure 1

Different transcripts of the GLB1 gene. (a) Scheme of the GLB1 gene. Different possible transcripts are shown. The primers used to amplify these transcripts are indicated by horizontal arrows. Black circles are natural stop codons whereas red circles indicate PTCs. (b) &(c) RT-PCR on cDNA from cycloheximide-treated (CHX) or not treated (NT) HeLa cells and human fibroblasts. The transcripts corresponding to each band are outlined on the right. (b) PCR using C1F/EBPR as primers. (c) PCR using EBPF/C3R as primers. * indicates bands for possible transcripts including only exon 3 or only exon 4.

Figure 2

BX minigene: mutated splice sites and scores. (a) Scheme of the BX minigene construct. Exons: grey boxes. Introns: horizontal lines. Two lines cutting introns indicate that the whole intron was not cloned. The naturally occurring β-Gal (above the exons) and EBP (below the exons) splicing pathways are indicated. * (in red) indicates the location of the changes introduced by site-directed mutagenesis in BX3 and BX4 constructs. Arrows indicate the location of T7 and SP6 primers. PCMV: CMV promotor; BGHpA: BGH polyadenylation site. (b) 3'ss wild-type and mutated sequences and calculated splicing scores. Changes introduced by mutagenesis are underlined. cons: plasmid construct.

Figure 3

Semiquantitative RT-PCR on cDNA from transfected HeLa cells with plasmids BX, BX3 and BX4. (a) Semiquantitative RT-PCR using T7/EBPR primers (left) and scheme of the transcript corresponding to each band is shown (right). (b) Percentage of transcripts including exon 3 versus transcripts lacking it, i.e. (1+2)/(3+4). (c) Percentage of transcripts including exon 4 versus transcripts lacking it, i.e. (1+3)/(2+4). Each value is the mean ± S.E. of two independent experiments, each of which was replicated three times. * when p < 0.05.

Figure 4

RT-PCR using primers T7/EBPR after cotransfection with the BX and SR plasmids. (a) Semiquantitative RT-PCR, using primers T7/EBPR, on cDNA from HeLa cells cotransfected with the BX plasmid together with a plasmid expressing one of the following proteins: SF2/ASF, SRp20, SRp40, SRp55, 9G8 and hnRNPA1. The BX lane is the control. The transcripts corresponding to each band are drawn on the right. (b) &(c) Quantification of radio-labelled RT-PCR for the different transcripts (b for transcripts including exon 3 versus transcripts lacking it, i.e. (1+2)/(3+4) and c for transcripts including exon 4 versus transcripts lacking it, i.e. (1+3)/(2+4)), considering BX transfection as 100% in each case. Each value is the mean ± S.E. of two independent experiments, each of which was replicated three times. * when p < 0.05.

Figure 5

RT-PCR using primers 2_4F/SP6 after cotransfection with the BX and SR plasmids. RT-PCR, using primers 2_4F and SP6, on cDNA from HeLa cells cotransfected with the BX plasmid together with a plasmid expressing one of the following proteins: ASF/SF2, SRp20, SRp40, SRp55, 9G8 and hnRNPA1. The transcripts corresponding to each band are drawn on the right.