Stop codons affect 5' splice site selection by surveillance of splicing

Abstract

Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5' SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5' SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs.

Figure 1

An intronic latent 5′ SS is activated in CAD mutant mini-genes devoid of upstream stop codons—RT-PCR analysis. (a) Schematic drawings of wild-type CAD mini-genes (open boxes, exons; heavy line, intron; narrow box, intronic sequence included as part of the exon in the latent RNA). The normal and latent 5′ SSs and the in-frame stop codons between them are indicated (S# designates the T nucleotide in a stop codon and its respective distance from the normal 5′ SS). (b and c) Gel electrophoretic analysis of RT-PCR DNA fragments obtained from CAD mini-genes. Sequences of stop codons and their mutations (underlined nucleotides) in each construct are indicated above each of the respective lanes. Also indicated (−/+) is the expected occurrence of latent splicing. Bands corresponding to precursor and mature (normal and latent) CAD fragments amplified with primers a+b (b) and primers c+b (c) are indicated by schematic drawings on the right. These assignments were confirmed by sequence analyses of the DNA fragments extracted from the gel. An additional minor band, which is assigned to a heteroduplex between precursor and mature PCR-amplified DNAs, occasionally appeared just below the 428-nt band in Figs. 1b, 2a, 3a, and 4a, as confirmed by sequence analyses of the DNA extracted from the gel, and by rerunning it on a second gel. Lane 2, control with untransfected cells. Lane 1, size markers, pBR322 cut with MspI.

Figure 2

Splicing at a latent 5′ SS in CAD pre-mRNA is suppressed by all three in-frame stop codon variants. RT-PCR analyses of CAD constructs were performed with primers a and b (a) and primers c and b (b). Lettering and symbols are as in Fig. 1.

Figure 3

An intronic latent 5′ SS is activated in frame-shifted CAD mutants that eliminate in-frame stop codons. S86 in CAD2 was frame-shifted by an AT insertion at a distance of 53 nt downstream of the normal 5′ SS (Mut 6 and Mut 7). All four stop codons in CAD1 (see Fig. 1b, CAD1) were mutated by a T insertion into S25, thus frame-shifting the remaining three (Mut 9). C, Control RNA from untransfected cells. RT-PCR analyses were performed with primers a and b (a) and primers c and b (b). Lettering and symbols are as in Fig. 1.

Figure 4

Abrogation of NMD does not reveal latent splicing. (a) Human 293T cells (lanes 1–13) were transfected with CAD mini-gene constructs as indicated (lanes 1–4 are cotransfections with pEGFP-N3). Twenty-four hours posttransfection the cultures were treated with CHX (20 μg/ml) for the indicated lengths of time. RNA was analyzed by RT-PCR as in Fig. 1b (lettering and symbols are as in Fig. 1). (b) Human 293T cells were cotransfected as indicated (β-globin WT, wild-type β-globin; β-globin Ter39, a mutant construct expressing β-globin mRNA having a PTC at position 39; CAD Ter, a mutant construct expressing CAD mRNA having a PTC in exon N+1). Treatment with CHX was as in a. β-globin mRNA was revealed by RT-PCR. Mature CAD 1 and CAD Ter mRNAs were revealed as in Fig. 1b.

Figure 5

An intronic latent 5′ SS is activated in IDUA mutant mini-genes devoid of upstream in-frame stop codons. (a) A schematic drawing of wild-type IDUA mini-genes (symbols are as in Fig. 1; S, intronic in-frame stop codon). (b and c) Gel electrophoretic analysis of RT-PCR DNA fragments obtained from IDUA mini-genes. The sequences of the normal 5′ SSs in the wild-type and the mutant mini-genes (mutations underlined) are indicated above each of the respective lanes. Bands corresponding to precursor and mature (normal and latent) IDUA fragments amplified with primers b+c (b) and primers d+c (c) are indicated by schematic drawings on the right (other symbols are as in Fig. 1). These assignments were confirmed by sequence analyses of the DNA fragments extracted from the gel. The additional minor band that appears just below the 537-nt band in lanes 4–6 of c was assigned to a heteroduplex between precursor and spliced PCR-amplified DNAs, as confirmed by sequence analyses of the DNA extracted from the gel, and by rerunning it on a second gel. Lane 2, control with RNA from untransfected cells. Lane 1, size markers, pBR322 cut with MspI.