An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon

Abstract

Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat alpha-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when "prespliced" to exon 2 it follows a "zero length exon" splicing pathway in which a newly generated 5' splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD.

FIG. 1.

TM pseudo-exon. (A) Exons 2 and 3 of the TM gene are mutually exclusive. Exon 3 is selected in most cells, but in smooth muscle cells exon 3 is repressed leading to exon 2 inclusion. This repression is mediated by a number of regulatory elements, including a pyrimidine tract, referred to as DY, located 170 to 225 nt downstream of exon 3 (black rectangle). Just downstream of DY is the apparent pseudo-exon characterized in the present study (P). (B) Multiple sequence alignment (CLUSTAL; asterisks denote fully conserved positions) of intronic sequences reveals that conservation extends beyond the DY pyrimidine tract (in boldface in the rat sequence) for another 120 nt. This conserved region encompasses a suboptimal GAG 3′ss, immediately followed by a 5′ss in a “zero-length exon” arrangement (in boldface). Toward the end of the conserved region is a second 5′ss (AAG|GT[C/T]TGT in boldface) defining a pseudo-exon, which is 107 nt in the rat. In all species, inclusion of the pseudo-exon to either exon 2 or 3 would introduce in-frame stop codons (boldface TGA) leading to NMD.

FIG. 2.

Dual-function ZLE and pseudo-exon. (A) Endogenous splicing intermediates were detected by nonquantitative RT-PCR from rat skeletal muscle (S), aorta (A), and uterus (U) RNA. Primers used to detect 2-P and 3-P products are indicated above. Products of pseudo-exon splicing to exon 2 (lanes 1 to 3) and exon 3 (lanes 4 to 6) and unspliced RNA are labeled alongside the gel. The asterisk indicates a PCR artifact that appears to arise by amplification of unspliced RNA with a subsequent 423-bp deletion between repeated GTCC motifs probably arising by template slippage. The upper band in lane 3 did not appear reproducibly and has not been identified. Lane M, size markers (100, 200, 300, 400, and 500 bp); lanes “−,” PCR no-template control. (B) Tropomyosin reporter constructs. pTS23D is wild type, and 2-P and 3-P have exons 2 and 3, respectively, “prespliced” to the ZLE/pseudo-exon. The observed splicing patterns of these constructs are indicated by the dashed lines. (C) Constructs were transfected into HeLa cells, and splicing was analyzed by RT-PCR with the primers SV5′2 and TM4. Spliced products are indicated schematically at the side, and the splice patterns are summarized in panel B).

FIG. 3.

Pseudo-exon splicing to exon 4 leading to NMD. (A) Rat smooth muscle (PAC-1) cells were incubated in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of puromycin. Pseudo-exon- to exon 4-spliced products were detected by semiquantitative RT-PCR with primer 107F in the pseudo-exon and primer 4R in exon 4 (lanes 3 and 4). RNA input levels were normalized by using beta-actin PCRs (lanes 1 and 2), indicating a threefold increase in P-4 product upon puromycin treatment. An increase of 3.2-fold was observed in an independent repeat. The effectiveness of the puromycin treatment was also monitored by observing the levels of PTB exon 11 skipping (lanes 5 and 6). The exon 11 skipped product trPTB has been shown to accumulate to 20-fold when the NMD machinery was inactivated by Upf-1 knockdown by RNAi (43). PCR primers were in PTB exons 8 and 12, so the products of exon 9 inclusion (PTB4) and skipping (PTB1) are also evident. The levels of exon 11-skipped products (trPTB1 and 4) as a proportion of total PTB products increased by 3.8-fold upon puromycin treatment and by 4.5-fold in an independent repeat. (B) Detection of pseudo-exon usage in various rat tissues. RT-PCR was carried out with primer 107F in the pseudo-exon and primer 4R in exon 4 with RNA from rat brain (B), gut (G), heart (H), liver (L), kidney (K), skeletal muscle (S), and cardiac myocytes (CM) (lanes 1 to 7, respectively). Pseudo-exon products were highest in heart (H), skeletal muscle (S), and cardiac myocytes (CM). RNA input levels were checked by using GAPDH PCRs (lower panel).

FIG. 4.

The pseudo-exon is easily activated by mutations. Various mutations were generated in the tropomyosin reporter construct pTS23D and mutant constructs transfected into HeLa cells. Splicing was analyzed by RT-PCR with primers SV5′2 and TM4 unless otherwise stated. (A) 3′ss mutants. Lane 1, GAG (wild type); lane 2, CAG; lane 3, AAG; lane 4, TAG; lane 5, no PCR template. Exon-specific PCRs were also carried out for the GAG (wild type) and CAG mutant using forward primers in exon 3 (lanes 6 to 8) or exon 2 (lanes 9 to 11). (B) Mutations generating AG dinucleotides 3 and 6 nt upstream of the pseudo-exon. Lane 1, wild type; lane 2, −6 TAG; lane 3, −3 TAG; lane 4, no template control. (C) Mutation of the ZLE 5′ss. The wild-type (wt) and mutant 5′ss sequences are shown. (D) Effect of spacers between the ZLE 3′ and 5′ splice sites. A 6-nt spacer was introduced into the ZLE in the pTS23D construct to generate the +6 construct. The constructs +6G, +9, and + 6G4 were derived from the +6 construct as indicated. (E) Effect of single base insertion mutations +12C (lane 2) and +12G (lane 3).

FIG. 5.

The pseudo-exon is coregulated with exon 3. (A) Levels of pseudo-exon splicing in PAC-1 smooth muscle cells and HeLa cells transfected with the wild-type pTS23D (lanes 5, 7, and 9) and +6 (lanes 6, 8, and 10) constructs. Endogenous TM exon 2 inclusion in PAC-1 cells was determined by RT-PCR with TM1 and TM4 primers, followed by digestion with PvuII (lanes 2 and 4) which cuts in exon 3. PAC-1 cells include exon 2 to different levels depending on the differentiation status of the cells (compare lanes 2 and 4). HeLa cells do not express TM, so no panel is shown (N/A), but they splice constructs almost exclusively to include exon 3. Construct RNA was analyzed by RT-PCR with SV5′2 and TM4 primers (lanes 5 to 10). Lanes 5 and 6 result from transfection of the same cells shown in lanes 1 and 2, while lanes 7 and 8 are transfections of the same cells shown in lanes 3 and 4. (B) HeLa cells were transfected with 0.5 μg of wild-type pTS23D (lane 1) or +6 construct (lane 2) and cotransfected with 0.5 μg of pGem4Z (lanes 1 and 2) or plasmids expressing hnRNPG (lane 3), hnRNPA1 (lane 4), hnRNPC (lane 5), Raver1 (lane 6), and PTB (lane 7).

FIG. 6.

The pseudo-exon has the profile of a real exon. A scatter plot of ESS (y axis) and ESE (x axis) scores for 462 pseudo-exons (red crosses) and 502 noncoding exons (green crosses) was prepared (44). A randomness of 0.1 to 0.3 was introduced for the points at zero to reduce overlap. The white areas running immediately parallel to the axes are because the ESE/ESS score can be zero or between 1 and n. Pseudo-exons from the human TM (box 1), growth hormone (box 2), ATM (box 3), chimp TM (box 4), mouse TM (box 5), rat TM (box 6), and dog TM (box 7) are indicated. The ESE and ESS counts for these test pseudo-exons are shown in the table. Note that the TM pseudo-exons from all species lie in an area that is highly enriched for genuine exons.